bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Impact of a Pesticide Treatment on Bacterial Soil Communities by Metabolic and Genetic Fingerprinting in Addition to Conventional Testing Procedures
Authors: Engelen Bert , Meinken Kristin , von Wintzingerode Friedrich , Heuer Holger , Malkomes Hans-Peter , Backhaus Horst
Published: December 2019

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of “fingerprinting” methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate
t
test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.
One Hundred Seventy-Fold Increase in Excretion of an FV Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-α) from
Escherichia coli
Caused by the Synergistic Effects of Glycine and Triton X-100
Authors: Yang Junbao , Moyana Terence , MacKenzie Samuel , Xia Qun , Xiang Jim
Published: December 2019

To target tumor necrosis factor alpha (TNF-α) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene V
K
LV
H
–TNF-α, which encodes the secreted form of single-chain fusion protein sFV/TNF-α in
Escherichia coli
. sFV/TNF-α was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30°C and 37 μmol of isopropyl-β-
d
-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37°C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-α was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-α in
E. coli
by using glycine and Triton X-100.
Genetics of Vegetative Incompatibility in
Cryphonectria parasitica
Authors: Cortesi Paolo , Milgroom Michael G.
Published: December 2019

Vegetative incompatibility in the chestnut blight fungus,
Cryphonectria parasitica
, in Europe is controlled by six unlinked
vic
loci, each with two alleles. Four previously identified
vic
loci (
vic1
,
vic2
,
vic3
, and
vic4
) were polymorphic in European vegetative compatibility (vc) types. Two new loci,
vic6
and
vic7
, also were identified among European vc types. In one cross,
vic
genes segregated independently at five loci, and 194 progeny were assigned to 32 vc types; none of these loci were linked. A total of 64 vc types were identified from all crosses. All 64 genotypes possible from six
vic
loci, each with two alleles (2
6
= 64), were identified and assigned to vc types. Based on our model, vc types v-c 5 and v-c 10, which had been used in previous genetic studies, differ by only five
vic
genes. Future studies of vc types in
C. parasitica
can use knowledge of
vic
genotypes for analysis of population genetic structure based on
vic
allele frequencies and to determine the effect of each
vic
gene on virus transmission between vc types.
Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/
o
-Xylene Monooxygenase from
Pseudomonas stutzeri
OX1
Authors: Chauhan Sadhana , Barbieri Paola , Wood Thomas K.
Published: December 2019

Toluene/
o
-xylene monooxygenase (ToMO) from
Pseudomonas stutzeri
OX1, which oxidizes toluene and
o
-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE),
cis
-1,2-DCE,
trans
-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol.
Escherichia coli
JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the
lac
promoter degraded TCE (3.3 μM), 1,1-DCE (1.25 μM), and chloroform (6.3 μM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min · mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl
−
atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.
Fungal Biotransformation of 6-Nitrochrysene
Authors: Pothuluri Jairaj V. , Sutherland John B. , Freeman James P. , Cerniglia Carl E.
Published: December 2019

The fungus
Cunninghamella elegans
was used to biotransform 6-nitrochrysene, a mutagen that is a widespread environmental contaminant. After 6 days, 74% of the
3
H-labeled 6-nitrochrysene added had been metabolized to two isomeric sulfate conjugates. These conjugates were separated by high-performance liquid chromatography and identified by UV-visible,
1
H nuclear magnetic resonance, and mass spectral techniques as 6-nitrochrysene 1-sulfate and 6-nitrochrysene 2-sulfate.
Effect of Humic Fractions and Clay on Biodegradation of Phenanthrene by a
Pseudomonas fluorescens
Strain Isolated from Soil
Authors: Ortega-Calvo Jose-Julio , Saiz-Jimenez Cesareo
Published: December 2019

The mineralization of phenanthrene in pure cultures of a
Pseudomonas fluorescens
strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.
Signal Transduction in the Protozoan Host
Hartmannella vermiformis
upon Attachment and Invasion by
Legionella micdadei
Authors: Abu Kwaik Yousef , Venkataraman Chandrasekar , Harb Omar S. , Gao Liang-Yong
Published: December 2019

The intracellular pathogens
Legionella micdadei
and
Legionella pneumophila
are the two most common
Legionella
species that cause Legionnaires’ disease. Intracellular replication within pulmonary cells is the hallmark of Legionnaires’ disease. In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires’ disease. In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host
Hartmannella vermiformis
by
L. micdadei
. Bacterial attachment prior to invasion of
H. vermiformis
by
L. micdadei
is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein. We have previously shown that this 170-kDa protein is the galactose
N
-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of
H. vermiformis
by
L. pneumophila
. Subsequent bacterial entry targets
L. micdadei
into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER). In contrast, uptake of
L. pneumophila
mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome. These results indicate that despite similarities in the
L. micdadei
and
L. pneumophila
attachment-mediated signal transduction mechanisms in
H. vermiformis
, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host.
Cloning, Nucleotide Sequence, and Expression in
Escherichia coli
of Levansucrase Genes from the Plant Pathogens
Pseudomonas syringae
pv. glycinea and
P. syringae
pv. phaseolicola
Authors: Hettwer Ursula , Jaeckel Frank R. , Boch Jens , Meyer Manfred , Rudolph Klaus , Ullrich Matthias S.
Published: December 2019

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in
Pseudomonas syringae
,
Erwinia amylovora
, and other bacterial species. The
lsc
genes encoding levansucrase from
P. syringae
pv. glycinea PG4180 and
P. syringae
pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the
lsc
gene in
Escherichia coli
was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3.0-kb
Pst
I fragment common to all six clones conferred levan synthesis on
E. coli
when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated
lsc
. Both ORFs showed high homology to the
E. amylovora
and
Zymomonas mobilis lsc
genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of
E. coli
harboring the
lsc
gene. Expression of
lsc
was found to be dependent on the vector-based P

lac

promoter, indicating that the native promoter of
lsc
was not functional in
E. coli
. Insertion of an antibiotic resistance cassette in the
lsc
gene abolished levan synthesis in
E. coli
. A PCR screening with primers derived from
lsc
of
P. syringae
pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.
Characterization of the Bactericidal Effects of Sodium Nitroprusside and Other Pentacyanonitrosyl Complexes on the Food Spoilage BacteriumClostridium sporogenes
Authors: Joannou Christopher L. , Cui Xiao-Yuan , Rogers Nicola , Vielotte Natasha , Torres Martinez Claudia L. , Vugman Ney V. , Hughes Martin N. , Cammack Richard
Published: December 2019

The potent bactericidal activity of sodium nitroprusside {SNP; Na2[Fe(CN)5(NO)]} towardsClostridium sporogeneshas been investigated. SNP inhibited cell growth in the concentration range of 10 to 40 μM. Concentrations above 80 μM caused irreversible loss of cell viability and cell lysis. Inhibition of cell growth was similar in complex and in defined media. SNP was found to be unreactive towards individual components of the defined medium, with the exception of cysteine. The chemical characteristics responsible for the potency of SNP were investigated by synthesizing analogs of SNP in which the Fe was replaced by different metals. The inhibitory potency of the pentacyanonitrosyl complexes decreased in the order Fe > Cr > V, which correlates with N-O stretching frequency (vNO). In contrast, the Ru complex which had avNO comparable to that of Fe was a poor inhibitor. Electron paramagnetic resonance spectroscopy showed that SNP was rapidly reduced to the paramagnetic Fe(I) compound [Fe(CN)4(NO)]2−on contact with cells. Analysis of fractions from SNP-treated cells showed 90% oxidation of thiols in the cell walls compared with those in control cells. The toxicity of SNP involves S-nitrosation and reduction, the lack of toxicity of the Ru analog being consistent with the fact that it has poor reactivity towards thiols. WhenC. sporogenescells were exposed to sublethal concentrations of SNP and viewed under the electron microscope, they showed blisters on the surface. These results point to the cell wall surface as a primary point of attack of the nitrosyl complex.
Contribution of Indole-3-Acetic Acid Production to the Epiphytic Fitness of
Erwinia herbicola
Authors: Brandl M. T. , Lindow S. E.
Published: December 2019

Erwinia herbicola
299R produces large quantities of indole-3-acetic acid (IAA) in culture media supplemented with
l
-tryptophan. To assess the contribution of IAA production to epiphytic fitness, the population dynamics of the wild-type strain and an IAA-deficient mutant of this strain on leaves were studied. Strain 299XYLE, an isogenic IAA-deficient mutant of strain 299R, was constructed by insertional interruption of the indolepyruvate decarboxylase gene of strain 299R with the
xylE
gene, which encodes a 2,3-catechol dioxygenase from
Pseudomonas putida
mt-2. The
xylE
gene provided a useful marker for monitoring populations of the IAA-deficient mutant strain in mixed populations with the parental strain in ecological studies. A root bioassay for IAA, in which strain 299XYLE inhibited significantly less root elongation than strain 299R, provided evidence that
E. herbicola
produces IAA on plant surfaces in amounts sufficient to affect the physiology of its host and that IAA production in strain 299R is not solely an in vitro phenomenon. The epiphytic fitness of strains 299R and 299XYLE was evaluated in greenhouse and field studies by analysis of changes in the ratio of the population sizes of these two strains after inoculation as mixtures onto plants. Populations of the parental strain increased to approximately twice those of the IAA-deficient mutant strain after coinoculation in a proportion of 1:1 onto bean plants in the greenhouse and onto pear flowers in field studies. In all experiments, the ratio of the population sizes of strain 299R and 299XYLE increased during periods of active growth on plant tissue but not when population sizes were not increasing with time.
Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria
Authors: Dias Benjamin , Weimer Bart
Published: December 2019

Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In
Lactococcus lactis
subsp.
cremoris
S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in
Brevibacterium linens
BL2, it possessed high levels of
l
-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.
Purification and Characterization of Three Thermostable Endochitinases of a Noble
Bacillus
Strain, MH-1, Isolated from Chitin-Containing Compost
Authors: Sakai Kenji , Yokota Akira , Kurokawa Hajime , Wakayama Mamoru , Moriguchi Mitsuaki
Published: December 2019

A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-
O
-dimethyl)-β-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus
Bacillus
. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from
Bacillus
sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75°C) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)
6
], chitinases L and S produced (GlcNAc)
2
at the highest rate while chitinase M produced (GlcNAc)
3
at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)
2
. Chitinase L produced (GlcNAc)
2
and (GlcNAc)
3
in most abundance from 66 and 11% partially acetylated chitosan. The
p
-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)
2
, and those of chitinases M and S were highest toward pNP-(GlcNAc)
3
. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl
2
, and (GlcNAc)
2
inhibited the activities of all three enzymes, while MnCl
2
and CaCl
2
slightly activated all of the enzymes.
Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria
Authors: Chu Kung-Hui , Alvarez-Cohen Lisa
Published: December 2019

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains,
Methylosinus trichosporium
OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both
M. trichosporium
OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed,
M. trichosporium
OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.
Cellular Events Involved in Survival of Individual Arbuscular Mycorrhizal Symbionts Growing in the Absence of the Host
Authors: Logi Cable , Sbrana Cristiana , Giovannetti Manuela
Published: December 2019

A survival strategy operating in the absence of the host was shown in obligately biotrophic arbuscular mycorrhizal (AM) symbionts. When no host-derived signals from the surrounding environment were perceived by germinating spores, fungal hyphae underwent a programmed growth arrest and resource reallocation, allowing long-term maintenance of viability and host infection capability. The early stages of mycelial growth of AM fungi were studied by a combination of time-lapse and video-enhanced light microscopy, image analysis, and immunodetection, with the aim of acquiring knowledge of cell events leading to the arrest of mycelial growth. The time-course of growth arrest was resolved by precisely timing the growth rate and magnitude of the mycelium originating from individual spores of
Glomus caledonium
. Extensive mycelial growth was observed during the first 15 days; thereafter, fungal hyphae showed retraction of protoplasm from the tips, with formation of retraction septa separating viable from empty hyphal segments. This active process involved migration of nuclei and cellular organelles and appeared to be functional in the ability of the fungus to survive in the absence of a host. Immunodetection of cytoskeletal proteins, metabolic activity, and the retention of infectivity of germinated spores confirmed the developmental data. The highest amounts of tubulins were detected when hyphal growth had ceased but when retraction of protoplasm was most active. This was consistent with the role of the cytoskeleton during protoplasm retraction. Succinate dehydrogenase activity in hyphae proximal to the mother spore was still detectable in 6-month-old mycelium, which remained viable and able to form appressoria and produce symbiotic structures.
Composition and Susceptibility to Chlorhexidine of Multispecies Biofilms of Oral Bacteria
Authors: Pratten J. , Barnett P. , Wilson M.
Published: December 2019

Using a constant-depth film fermentor, we have grown a six-membered biofilm community with a bacterial composition similar to that found in supragingival dental plaque. Cryosectioning revealed the distribution of bacteria throughout the biofilm. Exposure to 0.2% chlorhexidine for up to 5 min had little effect on biofilm viability.
Diversity of
Frankia
Strains in Root Nodules of Plants from the Families Elaeagnaceae and Rhamnaceae
Authors: Clawson Michael L. , Carú Margarita , Benson David R.
Published: December 2019

Partial 16S ribosomal DNAs (rDNAs) were PCR amplified and sequenced from
Frankia
strains living in root nodules of plants belonging to the families Elaeagnaceae and Rhamnaceae, including
Colletia hystrix
,
Elaeagnus angustifolia
, an unidentified
Elaeagnus
sp.,
Talguenea quinquenervia
, and
Trevoa trinervis
. Nearly full-length 16S rDNAs were sequenced from strains of
Frankia
living in nodules of
Ceanothus americanus
,
C. hystrix
,
Coriaria arborea
, and
Trevoa trinervis
. Partial sequences also were obtained from
Frankia
strains isolated and cultured from the nodules of
C. hystrix
,
Discaria serratifolia
,
D. trinervis
,
Retanilla ephedra
,
T. quinquenervia
, and
T. trinervis
(Rhamnaceae). Comparison of these sequences and other published sequences of
Frankia
16S rDNA reveals that the microsymbionts and isolated strains from the two plant families form a distinct phylogenetic clade, except for those from
C. americanus
. All sequences in the clade have a common 2-base deletion compared with other
Frankia
strains. Sequences from
C. americanus
nodules lack the deletion and cluster with
Frankia
strains infecting plants of the family Rosaceae. Published plant phylogenies (based on chloroplast
rbcL
sequences) group the members of the families Elaeagnaceae and Rhamnaceae together in the same clade. Thus, with the exception of
C. americanus
, actinorhizal plants of these families and their
Frankia
microsymbionts share a common symbiotic origin.
Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene,
xylA
, from
Aspergillus oryzae
KBN616
Authors: Kitamoto Noriyuki , Yoshino Shoko , Ohmiya Kunio , Tsukagoshi Norihiro
Published: December 2019

β-Xylosidase secreted by the shoyu koji mold,
Aspergillus oryzae
, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of
A. oryzae
KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The
xylA
gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to
Aspergillus nidulans
XlnD (70%),
Aspergillus niger
XlnD (64%), and
Trichoderma reesei
BxII (63%). The
xylA
gene was overexpressed under control of the strong and constitutive
A. oryzae TEF1
promoter. One of the
A. oryzae
transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense
xylA
gene expressed under control of the
A. oryzae TEF1
promoter decreased the β-xylosidase level in
A. oryzae
to about 20% of that of the host strain.
Nitrogen Cycling and Community Structure of Proteobacterial β-Subgroup Ammonia-Oxidizing Bacteria within Polluted Marine Fish Farm Sediments
Authors: McCaig Allison E. , Phillips Carol J. , Stephen John R. , Kowalchuk George A. , Harvey S. Martyn , Herbert Rodney A. , Embley T. Martin , Prosser James I.
Published: December 2019

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial β-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine
Nitrosomonas
group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel
Nitrosomonas
subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution.
Phylogenetic Affinity of a Wide, Vacuolate, Nitrate-Accumulating
Beggiatoa
sp. from Monterey Canyon, California, with
Thioploca
spp
Authors: Ahmad Azeem , Barry James P. , Nelson Douglas C.
Published: December 2019

Environmentally dominant members of the genus
Beggiatoa
and
Thioploca
spp. are united by unique morphological and physiological adaptations (S. C. McHatton, J. P. Barry, H. W. Jannasch, and D. C. Nelson, Appl. Environ. Microbiol. 62:954–958, 1996). These adaptations include the presence of very wide filaments (width, 12 to 160 μm), the presence of a central vacuole comprising roughly 80% of the cellular biovolume, and the capacity to internally concentrate nitrate at levels ranging from 150 to 500 mM. Until recently, the genera
Beggiatoa
and
Thioploca
were recognized and differentiated on the basis of morphology alone; they were distinguished by the fact that numerous
Thioploca
filaments are contained within a common polysaccharide sheath, while
Beggiatoa
filaments occur singly. Vacuolate
Beggiatoa
or
Thioploca
spp. can dominate a variety of marine sediments, seeps, and vents, and it has been proposed (H. Fossing, V. A. Gallardo, B. B. Jorgensen, M. Huttel, L. P. Nielsen, H. Schulz, D. E. Canfield, S. Forster, R. N. Glud, J. K. Gundersen, J. Kuver, N. B. Ramsing, A. Teske, B. Thamdrup, and O. Ulloa, Nature [London] 374:713–715, 1995) that members of the genus
Thioploca
are responsible for a significant portion of total marine denitrification. In order to investigate the phylogeny of an environmentally dominant
Beggiatoa
sp., we analyzed complete 16S rRNA gene sequence data obtained from a natural population found in Monterey Canyon cold seeps. Restriction fragment length polymorphism analysis of a clone library revealed a dominant clone, which gave rise to a putative Monterey
Beggiatoa
16S rRNA sequence. Fluorescent in situ hybridization with a sequence-specific probe confirmed that this sequence originated from wide
Beggiatoa
filaments (width, 65 to 85 μm). A phylogenetic tree based on evolutionary distances indicated that the Monterey
Beggiatoa
sp. falls in the gamma subdivision of the class
Proteobacteria
and is most closely related to the genus
Thioploca
. This vacuolate
Beggiatoa—Thioploca
cluster and a more distantly related freshwater
Beggiatoa
species cluster form a distinct phylogenetic group.
Conditions for Vigorous Growth on Sulfide and Reactor-Scale Cultivation Protocols for the Thermophilic Green Sulfur Bacterium
Chlorobium tepidum
Authors: Mukhopadhyay Biswarup , Johnson Eric F. , Ascano Manuel
Published: December 2019

We describe a reactor-scale cultivation protocol for the fastest-growing and only known thermophilic member of the family
Chlorobiaceae
,
Chlorobium tepidum
. We discovered that
C. tepidum
would grow with sulfide as the sole electron source at rates and with final cell yields comparable to those found with thiosulfate only if the sulfide concentration was maintained below 0.1 mM and the culture redox potential was at −300 ± 20 mV. Such was also the requirement for growth in a photobioreactor when thiosulfate (optimum level, 12 mM) was used as the preferred electron source. For cultivation of
C. tepidum
on a 5- to 500-ml scale, we used the system of Balch and Wolfe (Appl. Environ. Microbiol. 32:781–791, 1976) using stopper-sealed serum tubes and bottles as an alternative to the methods commonly used for the cultivation of phototrophic anaerobes and obtained consistent results.
Symbiotic Deficiencies Associated with a
coxWXYZ
Mutant of
Bradyrhizobium japonicum
Authors: Surpin Marci Ann , Maier Robert J.
Published: December 2019

The terminal oxidase complexes encoded by
coxMNOP
and
coxWXYZ
were studied by analysis of mutations in each of the two oxidases. Carbon monoxide difference spectra obtained from membranes of
coxMNOP
mutant bacteroids were like those obtained for the wild type, whereas bacteroid membranes of a
coxWXYZ
mutant were deficient in CO-reactive cytochrome
b
. Experiments involving cyanide inhibition of oxidase activity were consistent with the conclusion that the
coxX
mutant is deficient in a membrane-associated O
2
-binding component. The viable cell number (bacteria that could be recultured from crushed nodules) was 20 to 29% lower for the
coxX
mutant than for the wild-type or the CoxN
−
strain. In three separate greenhouse studies, nodules of a
coxX
mutant had significantly lower (28 to 34% less) acetylene reduction rates than the wild-type nodules did, and plants inoculated with a double mutant (
coxMNOP coxWZYZ
) had rates 30% lower than those of wild-type-inoculated plants.
Characterization of a Facultatively Psychrophilic Bacterium,
Vibrio rumoiensis
sp. nov., That Exhibits High Catalase Activity
Authors: Yumoto Isao , Iwata Hideaki , Sawabe Tomoo , Ueno Keisuke , Ichise Nobutoshi , Matsuyama Hidetoshi , Okuyama Hidetoshi , Kawasaki Kosei
Published: December 2019

A novel facultatively psychrophilic bacterium, strain S-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses H
2
O
2
as a bleaching and microbicidal agent. The catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of
Corynebacterium glutamicum
,
Staphylococcus aureus
,
Pseudomonas fluorescens
, and five other species tested in this study. The strain seemed to possess only one kind of catalase, according to the results of polyacrylamide gel electrophoresis of the cell extract. The optimum temperature for catalase activity was about 30°C, which was about 20°C lower than that for bovine catalase activity. Electron microscopic observation revealed that the surface of the microorganism was covered by blebs. Although the isolate was nonflagellated, its taxonomic position on the basis of physiological and biochemical characteristics and analysis of 16S rRNA sequence and DNA-DNA relatedness data indicated that strain S-1 is a new species belonging to the genus
Vibrio
. Accordingly, we propose the name
Vibrio rumoiensis
. The type strain is S-1 (FERM P-14531).
Cloning and Analysis of the
l
-Lactate Utilization Genes from
Streptococcus iniae
Authors: Gibello A. , Collins M. D. , Domínguez L. , Fernández-Garayzábal J. F. , Richardson P. T.
Published: December 2019

The presence of lactate oxidase was examined in eight
Streptococcus
species and some related species of bacteria. A clone (pGR002) was isolated from a genomic library of
Streptococcus iniae
generated in
Escherichia coli
, containing a DNA fragment spanning two genes designated
lctO
and
lctP
. We show that these genes are likely to be involved in the
l
-lactic acid aerobic metabolism of this organism. This DNA fragment has been sequenced and characterized. A comparison of the deduced amino acid sequence of LctP protein demonstrated that the protein had significant homology with the
l
-lactate permeases of other bacteria. The amino acid sequence of the LctO protein of
S. iniae
also showed a strong homology to
l
-lactate oxidase from
Aerococcus viridans
and some NAD-independent lactate dehydrogenases, all belonging to the family of flavin mononucleotide-dependent α-hydroxyacid-oxidizing enzymes. Biochemical assays of the gene products confirm the identity of the genes from the isolated DNA fragment and reveal a possible role for the lactate oxidase from
S. iniae
. This lactate oxidase is discussed in relation to the growth of the organism in response to carbon source availability.
Identification of
Bacillus thuringiensis
Delta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity
Authors: de Maagd Ruud A. , Bakker Petra , Staykov Nikola , Dukiandjiev Stefan , Stiekema Willem , Bosch Dirk
Published: December 2019

Cry1C domain III amino acid residues involved in specificity for beet armyworm (
Spodoptera exigua
) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against
S. exigua
. Block E was also required for optimal activity against the tobacco hornworm (
Manduca sexta
), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full
S. exigua
activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against
S. exigua
but resulted in higher activity against
M. sexta
. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved in
S. exigua
-specific activity as well as sometimes involved in
M. sexta
-specific activity.
Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments
Authors: Vetriani Costantino , Jannasch Holger W. , MacGregor Barbara J. , Stahl David A. , Reysenbach Anna-Louise
Published: December 2019

During the past few years
Archaea
have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of
Archaea
in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms,
Crenarchaeota
and
Euryarchaeota
. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.
Use of the [14C]Leucine Incorporation Technique To Measure Bacterial Production in River Sediments and the Epiphyton
Authors: Fischer Helmut , Pusch Martin
Published: December 2019

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [14C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 μM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 μM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.
Regulation of Peroxidase Transcript Levels in Liquid Cultures of the Ligninolytic Fungus
Pleurotus eryngii
Authors: Ruiz-Dueñas Francisco Javier , Guillén Francisco , Camarero Susana , Pérez-Boada Marta , Martínez María Jesús , Martínez Ángel T.
Published: December 2019

A versatile peroxidase able to oxidize Mn
2+
as well as phenolic and nonphenolic aromatic compounds is produced in peptone-containing liquid cultures of
Pleurotus eryngii
encoded by the gene
mnpl
. The regulation of its transcript levels was investigated by Northern blotting of total RNA. High-peroxidase transcripts and activity were found in cultures grown in glucose-peptone medium, whereas only basal levels were detected in glucose-ammonium medium. The addition of more than 25 μM Mn
2+
to the former medium did not result in detectable peroxidase transcripts or activity. Potential regulators were also added to isolated mycelium. In this way, it was shown that high transcript levels (in peroxidase-expressing mycelium) were maintained on peptone, whereas expression was not induced in short-term incubation experiments. Similar results were obtained with Mn
2+
ions. Strong induction of
mnpl
expression was caused by exogenous H
2
O
2
or by continuous H
2
O
2
generation during redox cycling of menadione. By the use of the latter system in the presence of Fe
3+
, which catalyzes the reduction of H
2
O
2
to hydroxyl radical, it was shown for the first time that the presence of this strong oxidant causes a rapid increase of the transcripts of a ligninolytic peroxidase. In conclusion, peptone and Mn
2+
affect the levels of transcripts of this versatile peroxidase in culture, and reduced oxygen species induce short-term expression in isolated mycelium, probably via a stress response mechanism.
Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by
Bacillus cereus
Authors: Dietrich R. , Fella C. , Strich S. , Märtlbauer E.
Published: December 2019

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by
Bacillus cereus
were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L
2
component, and antibody 1C2 was specific for the L
1
protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of
B. cereus
was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with
B. cereus
sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of
B. cereus
strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.
Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of
Acinetobacter
sp. Strain BD413
Authors: Busch Silke , Rosenplänter Christine , Averhoff Beate
Published: December 2019

Although the high level of competence for natural transformation of
Acinetobacter
sp. strain BD413 has been the subject of numerous studies, only two competence genes,
comC
and
comP
, have been identified to date. By chromosomal walking analysis we found two overlapping open reading frames, designated
comE
and
comF
, starting 61 bp downstream of
comC
.
comE
and
comF
are expressed as stable proteins in
Escherichia coli
, thus proving that they are indeed coding regions, but expression was successful only with 5′-deleted genes. ComE and ComF are similar to pilins and pilin-like components. Both genes were mutated, and the phenotypes of the mutants were analyzed. Natural transformation in
comF
mutants is 1,000-fold reduced, whereas
comE
mutants exhibit 10-fold-reduced transformation frequencies. This is clear evidence that
comE
and
comF
are involved in natural transformation. However, ComE and ComF are specific for DNA translocation, since
comE
and
comF
defects affected neither piliation nor lipase secretion. These results suggest that the type IV pili, the general protein secretion pathway, and the DNA translocation machinery in
Acinetobacter
sp. strain BD413 are evolutionary related but functionally distinct systems.
Green Fluorescent Protein-Marked
Pseudomonas fluorescens
: Localization, Viability, and Activity in the Natural Barley Rhizosphere
Authors: Normander Bo , Hendriksen Niels B. , Nybroe Ole
Published: December 2019

The
gfp
-tagged
Pseudomonas fluorescens
biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.