bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of Two Forms of α-Amylases and Structural Implication
Authors: Ohdan Kohji , Kuriki Takashi , Kaneko Hiroki , Shimada Jiro , Takada Toshikazu , Fujimoto Zui , Mizuno Hiroshi , Okada Shigetaka
Published: December 2019

Complete (Ba-L) and truncated (Ba-S) forms of α-amylases from
Bacillus subtilis
X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of
B. subtilis
X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.
Unexpected Population Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia
Authors: Minz Dror , Fishbain Susan , Green Stefan J. , Muyzer Gerard , Cohen Yehuda , Rittmann Bruce E. , Stahl David A.
Published: December 2019

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-μm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families “Desulfovibrionaceae” and “Desulfobacteriaceae,”Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.
Dephosphorylation of Phytate by Using the
Aspergillus niger
Phytase with a High Affinity for Phytate
Authors: Nagashima Tadashi , Tange Tatsuya , Anazawa Hideharu
Published: December 2019

A phytase (EC
3.1.3.8
) with a high affinity for phytic acid was found in
Aspergillus niger
SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 ± 4.6 μM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low

K
m

phytase from
A. niger
SK-57 and a high

K
m

phytase from
Aspergillus ficuum
was recognized.
Antiproliferative Effects of Homogenates Derived from Five Strains of Candidate Probiotic Bacteria
Authors: Pessi Tanja , Sütas Yelda , Saxelin Maija , Kallioinen Harri , Isolauri Erika
Published: December 2019

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including
Lactobacillus rhamnosus
GG,
Bifidobacterium lactis
,
Lactobacillus acidophilus
,
Lactobacillus delbrueckii
subsp.
bulgaricus
, and
Streptococcus thermophilus
. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of
L. rhamnosus
GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.
Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut
Authors: Suau Antonia , Bonnet Régis , Sutren Malène , Godon Jean-Jacques , Gibson Glenn R. , Collins Matthew D. , Doré Joel
Published: December 2019

The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the
Bacteroides
group, the
Clostridium coccoides
group, and the
Clostridium leptum
subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g.,
Bacteroides thetaiotaomicron
,
Fusobacterium prausnitzii
, and
Eubacterium rectale
). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.
In Vitro Utilization of Amylopectin and High-Amylose Maize (Amylomaize) Starch Granules by Human Colonic Bacteria
Authors: Wang Xin , Conway Patricia Lynne , Brown Ian Lewis , Evans Anthony John
Published: December 2019

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that
Bifidobacterium
spp.,
Bacteroides
spp.,
Fusobacterium
spp., and strains of
Eubacterium
,
Clostridium
,
Streptococcus
, and
Propionibacterium
could hydrolyze the gelatinized amylopectin maize starch, while only
Bifidobacterium
spp. and
Clostridium butyricum
could efficiently utilize high-amylose maize starch granules. In fact,
C. butyricum
and
Bifidobacterium
spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on
Bifidobacterium
and
Bacteroides
cells and were extracellular for
C. butyricum
. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the
Bifidobacterium
cells produced several starch-degrading enzymes with high relative molecular (
M
r
) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that
Bifidobacterium
spp. and
C. butyricum
degraded and utilized granules of amylomaize starch.
Survival, Physiology, and Lysis of
Lactococcus lactis
in the Digestive Tract
Authors: Drouault Sophie , Corthier Gérard , Ehrlich S. Dusko , Renault Pierre
Published: December 2019

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the
luxA-luxB
gene of
Vibrio harveyi
and the
gfp
gene of
Aequora victoria
, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of
Lactococcus lactis
in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.
Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom
Lentinula edodes
Authors: Zhao J. , Kwan H. S.
Published: December 2019

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom
Lentinula edodes
was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the
L. edodes lac1
gene, were isolated, and DNA sequence analysis demonstrated that
lac1
encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the
L. edodes
Lac1 proteins. A fragment of a second laccase gene (
lac2
) was also isolated, and competitive PCR showed that expression of
lac1
and
lac2
genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.
Stress Responses as a Tool To Detect and Characterize the Mode of Action of Antibacterial Agents
Authors: Bianchi Allison A. , Baneyx François
Published: December 2019

Single-copy gene fusions between the
lacZ
reporter gene and
Escherichia coli
strains containing promoters induced by cold shock (
cspA
), cytoplasmic stress (
ibp
), or protein misfolding in the cell envelope (P3
rpoH
) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the
cspA
::
lacZ
or
ibp
::
lacZ
gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3
rpoH
::
lacZ
and
ibp
::
lacZ
fusions, while the β-lactam antibacterial agent carbenicillin activated only the P3
rpoH
promoter. Nalidixic acid, a compound that causes DNA damage, downregulated β-galactosidase synthesis from P3
rpoH
but had little effect on expression of the reporter enzyme from either the
cspA
or
ibp
promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of
Streptomyces venezuelae
cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.
Characterization and Identification of Numerically Abundant Culturable Bacteria from the Anoxic Bulk Soil of Rice Paddy Microcosms
Authors: Chin Kuk-Jeong , Hahn Dittmar , Hengstmann Ulf , Liesack Werner , Janssen Peter H.
Published: December 2019

Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 × 10
8
cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4′,6-diamidino-2-phenylindole staining, was 4.8 × 10
8
cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division
Verrucomicrobia
, the
Cytophaga-Flavobacterium-Bacteroides
division, clostridial cluster XIVa, clostridial cluster IX,
Bacillus
spp., and the class
Actinobacteria
. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050–5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.
Gene Transfer in the Gastrointestinal Tract
Authors: Netherwood Trudy , Bowden R. , Harrison P. , O’Donnell A. G. , Parker D. S. , Gilbert H. J.
Published: December 2019

The maximum in vivo transfer rate of plasmid pAMβ1 in the gut was 0.03 transconjugant per recipient cell, and this rate could be simulated in vitro only by forced filter mating. Transfer was not detected in liquid culture matings. Our findings demonstrate that in vitro methods, such as forced filter mating and liquid mating, underestimate the in vivo rates of gene transfer.
Isolation from the
Sorghum bicolor
Mycorrhizosphere of a Bacterium Compatible with Arbuscular Mycorrhiza Development and Antagonistic towards Soilborne Fungal Pathogens
Authors: Budi S. W. , van Tuinen D. , Martinotti G. , Gianinazzi S.
Published: December 2019

A gram-positive bacterium with antagonistic activity towards soilborne fungal pathogens has been isolated from the mycorrhizosphere of
Sorghum bicolor
inoculated with
Glomus mosseae
. It has been identified as
Paenibacillus
sp. strain B2 based on its analytical profile index and on 16S ribosomal DNA analysis. Besides having antagonistic activity, this bacterium stimulates mycorrhization.
Cloning, Overexpression, and Mutagenesis of the
Sporobolomyces salmonicolor
AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (
S
)-4-Chloro-3-Hydroxybutanoate
Authors: Kita Keiko , Fukura Takanobu , Nakase Koh-Ichi , Okamoto Kenji , Yanase Hideshi , Kataoka Michihiko , Shimizu Sakayu
Published: December 2019

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in
Sporobolomyces salmonicolor
AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (
S
)-4-chloro-3-hydroxybutanoate. The
ARII
gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced in
Escherichia coli
about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed in
E. coli
was purified to homogeneity and had the same catalytic properties as ARII purified from
S. salmonicolor
. To examine the contribution of the dinucleotide-binding motif G
19
-X-X-G
22
-X-X-A
25
, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G
19
→A and G
22
→A mutant enzymes by 4-COBE did not occur. The A
25
→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.
Cloning and Characterization of an
Aspergillus nidulans
Gene Involved in the Regulation of Penicillin Biosynthesis
Authors: Van den Brulle Jan , Steidl Stefan , Brakhage Axel A.
Published: December 2019

To identify regulators of penicillin biosynthesis, a previously isolated mutant of
Aspergillus nidulans
(Prg-1) which carried the
trans
-acting
prgA1
mutation was used. This mutant also contained fusions of the penicillin biosynthesis genes
acvA
and
ipnA
with reporter genes (
acvA
-
uidA
and
ipnA
-
lacZ
) integrated in a double-copy arrangement at the chromosomal
argB
gene. The
prgA1
mutant strain exhibited only 20 to 50% of the
ipnA
-
lacZ
and
acvA
-
uidA
expression exhibited by the wild-type strain and had only 20 to 30% of the penicillin produced by the wild-type strain. Here, using complementation with a genomic cosmid library, we isolated a gene (
suAprgA1
) which complemented the
prgA1
phenotype to the wild-type phenotype; i.e., the levels of expression of both gene fusions and penicillin production were nearly wild-type levels. Analysis of the
suAprgA1
gene in the
prgA1
mutant did not reveal any mutation in the
suAprgA1
gene or unusual transcription of the gene. This suggested that the
suAprgA1
gene is a suppressor of the
prgA1
mutation. The
suAprgA1
gene is 1,245 bp long. Its five exons encode a deduced protein that is 303 amino acids long. The putative SUAPRGA1 protein was similar to both the human p32 protein and Mam33p of
Saccharomyces cerevisiae
. Analysis of the ordered gene library of
A. nidulans
indicated that
suAprgA1
is located on chromosome VI. Deletion of the
suAprgA1
gene resulted in an approximately 50% reduction in
ipnA
-
lacZ
expression and in a slight reduction in
acvA
-
uidA
expression. The Δ
suAprgA1
strain produced about 60% of the amount of penicillin produced by the wild-type strain.
Biosynthesis of Novel Exopolymers by
Aureobasidium pullulans
Authors: Lee Jin W. , Yeomans Walter G. , Allen Alfred L. , Deng Fang , Gross Richard A. , Kaplan David L.
Published: December 2019

Aureobasidium pullulans
ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 × 10
6
to 2.12 × 10
6
to 0.85 × 10
6
to 0.77 × 10
6
with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from 2.73 × 10
6
to 4.86 × 10
6
. There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.
Transformation of
Escherichia coli
with DNA from
Saccharomyces cerevisiae
Cell Lysates
Authors: Adam Ana Cristina , González-Blasco Gracia , Rubio-Texeira Marta , Polaina Julio
Published: December 2019

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of
Saccharomyces cerevisiae
to
Escherichia coli
. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform
E. coli
. Transfer of DNA was measured by determining the number of ampicillin-resistant
E. coli
clones. Our results show that transmission of the Amp
r
gene to
E. coli
by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per μg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.
Secretion of Cryparin, a Fungal Hydrophobin
Authors: McCabe Patricia M. , Van Alfen Neal K.
Published: December 2019

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete
Cryphonectria parasitica
. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of
C. parasitica
, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.
The Hemolytic Enterotoxin HBL Is Broadly Distributed among Species of the
Bacillus cereus
Group
Authors: Prüß Birgit M. , Dietrich Richard , Nibler Birgit , Märtlbauer Erwin , Scherer Siegfried
Published: December 2019

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the
Bacillus cereus
group with the exception of
Bacillus anthracis. hblA
, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including
B. cereus
and
Bacillus thuringiensis
strains and the other one consisting of
Bacillus mycoides
,
Bacillus pseudomycoides
, and
Bacillus weihenstephanensis
strains. From eight
B. thuringiensis
strains, the enterotoxin gene
hblA
could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L
1
, L
2
, and B components. Eleven of 16
B. mycoides
strains, all 3
B. pseudomyoides
strains, 9 of 15
B. weihenstephanensis
strains, and 10 of 23
B. cereus
strains carried
hblA
. While HBL was not expressed in the
B. pseudomycoides
strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the
B. cereus
group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.
Phototrophs in High-Iron-Concentration Microbial Mats: Physiological Ecology of Phototrophs in an Iron-Depositing Hot Spring
Authors: Pierson B. K. , Parenteau M. N. , Griffin B. M.
Published: December 2019

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [14C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.
Biodegradation of Free Phytol by Bacterial Communities Isolated from Marine Sediments under Aerobic and Denitrifying Conditions
Authors: Rontani Jean-François , Bonin Patricia C. , Volkman John K.
Published: December 2019

Biodegradation of (
E
)-phytol [3,7,11,15-tetramethylhexadec-2(
E
)-en-1-ol] by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20°C. This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10,14-trimethylpentadecan-2-one and (
E
)-phytenic acid. The first step in both aerobic and anaerobic bacterial degradation of (
E
)-phytol involves the transient production of (
E
)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one. Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations. Since (
E
)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15°C), the major part of (
E
)-phytol appeared to be biodegraded in situ via (
E
)-phytenic acid. (
Z
)- and (
E
)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core. This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating β-decarboxymethylation and β-oxidation reaction sequences induced by denitrifiers. Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (
E
)-phytol.
Determining Sources of Fecal Pollution in a Rural Virginia Watershed with Antibiotic Resistance Patterns in Fecal Streptococci
Authors: Hagedorn Charles , Robinson Sandra L. , Filtz Jennifer R. , Grubbs Sarah M. , Angier Theresa A. , Reneau Raymond B.
Published: December 2019

Nonpoint sources of pollution that contribute fecal bacteria to surface waters have proven difficult to identify. Knowledge of pollution sources could aid in restoration of the water quality, reduce the amounts of nutrients leaving watersheds, and reduce the danger of infectious disease resulting from exposure to contaminated waters. Patterns of antibiotic resistance in fecal streptococci were analyzed by discriminant and cluster analysis and used to identify sources of fecal pollution in a rural Virginia watershed. A database consisting of patterns from 7,058 fecal streptococcus isolates was first established from known human, livestock, and wildlife sources in Montgomery County, Va. Correct fecal streptococcus source identification averaged 87% for the entire database and ranged from 84% for deer isolates to 93% for human isolates. To field test the method and the database, a watershed improvement project (Page Brook) in Clarke County, Va., was initiated in 1996. Comparison of 892 known-source isolates from that watershed against the database resulted in an average correct classification rate of 88%. Combining all animal isolates increased correct classification rates to ≥95% for separations between animal and human sources. Stream samples from three collection sites were highly contaminated, and fecal streptococci from these sites were classified as being predominantly from cattle (>78% of isolates), with small proportions from waterfowl, deer, and unidentified sources (≈7% each). Based on these results, cattle access to the stream was restricted by installation of fencing and in-pasture watering stations. Fecal coliforms were reduced at the three sites by an average of 94%, from prefencing average populations of 15,900 per 100 ml to postfencing average populations of 960 per 100 ml. After fencing, <45% of fecal streptococcus isolates were classified as being from cattle. These results demonstrate that antibiotic resistance profiles in fecal streptococci can be used to reliably determine sources of fecal pollution, and water quality improvements can occur when efforts to address the identified sources are made.
Estimation of Methanogen Biomass by Quantitation of Coenzyme M
Authors: Elias Dwayne A. , Krumholz Lee R. , Tanner Ralph S. , Suflita Joseph M.
Published: December 2019

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363–370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 ± 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(
N
)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.
Polyphosphates in Intraradical and Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus,
Gigaspora margarita
Authors: Solaiman M. Zakaria , Ezawa Tatsuhiro , Kojima Tomoko , Saito Masanori
Published: December 2019

The amount of polyphosphate in the intraradical and extraradical hyphae of
Gigaspora margarita
was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.
Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in
Aspergillus parasiticus
Authors: Zhou Renqing , Linz John E.
Published: December 2019

The
nor-1
gene is involved in aflatoxin biosynthesis in
Aspergillus parasiticus
and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in
Escherichia coli
converted the 1′ keto group of norsolorinic acid to the 1′ hydroxyl group of averantin in crude
E. coli
cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.
Effect of O-Side-Chain-Lipopolysaccharide Chemistry on Metal Binding
Authors: Langley S. , Beveridge T. J.
Published: December 2019

Pseudomonas aeruginosa
PAO1 produces two chemically distinct types of lipopolysaccharides (LPSs), termed A-band LPS and B-band LPS. The A-band O-side chain is electroneutral at physiological pH, while the B-band O-side chain contains numerous negatively charged sites due to the presence of uronic acid residues in the repeat unit structure. Strain PAO1 (A
+
B
+
) and three isogenic LPS mutants (A
+
B
−
, A
−
B
+
, and A
−
B
−
) were studied to determine the contribution of the O-side-chain portion of LPS to metal binding by the surfaces of gram-negative cells. Transmission electron microscopy with energy-dispersive X-ray spectroscopy was used to locate and analyze sites of metal deposition, while atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry were used to perform bulk quantitation of bound metal. The results indicated that cells of all of the strains caused the precipitation of gold as intracellular, elemental crystals with a
d
-spacing of 2.43 Å. This type of precipitation has not been reported previously for gram-negative cells and suggests that in the organisms studied gold binding is not a surface-mediated event. All four strains bound similar amounts of copper (0.213 to 0.222 μmol/mg [dry weight] of cells) at the cell surface, suggesting that the major surface metal-binding sites reside in portions of the LPS which are common to all strains (perhaps the phosphoryl groups in the core-lipid A region). However, significant differences were observed in the abilities of strains dps89 (A
−
B
+
) and AK1401 (A
+
B
−
) to bind iron and lanthanum, respectively. Strain dps89 caused the precipitation of iron (1.623 μmol/mg [dry weight] of cells) as an amorphous mineral phase (possibly iron hydroxide) on the cell surface, while strain AK1401 nucleated precipitation of lanthanum (0.229 μmol/mg [dry weight] of cells) as apiculate, surface-associated crystals. Neither iron nor lanthanum precipitates were observed on the cells of other strains, which suggests that the combination of A-band LPS and B-band LPS produced by a cell may result in a cell surface which promotes the formation of metal-rich precipitates. We therefore propose that the negatively charged sites located in the O-side chains are not directly responsible for the binding of metallic ions; however, the B-band LPS molecule as a whole may contribute to overall cell surface properties which favor the precipitation of distinct metal-rich mineral phases.
Hydrolysis of Pork Muscle Sarcoplasmic Proteins by
Lactobacillus curvatus
and
Lactobacillus sake
Authors: Fadda Silvina , Sanz Yolanda , Vignolo Graciela , Aristoy M.-Concepción , Oliver Guillermo , Toldrá Fidel
Published: December 2019

Lactobacillus curvatus
CECT 904 and
Lactobacillus sake
CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when
L. curvatus
was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation.
L. sake
accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.
Functional Analyses of the Promoters in the Lantibiotic Mutacin II Biosynthetic Locus in
Streptococcus mutans
Authors: Qi Fengxia , Chen Ping , Caufield Page W.
Published: December 2019

The lantibiotic bacteriocin mutacin II is produced by the group II
Streptococcus mutans
. The mutacin II biosynthetic locus consists of seven genes,
mutR
, -
A
, -
M
, -
T
, -
F
, -
E
, and -
G
, organized as two operons. The
mutAMTFEG
operon is transcribed from the
mutA
promoter 55 bp upstream of the translation start codon for MutA, while the
mutR
promoter is 76 bp upstream of the
mutR
structural gene. Expression of the
mutA
promoter is regulated by the components of the growth medium, while the
mutR
promoter activity does not seem to be affected by these conditions. Inactivation of
mutR
abolishes transcription of the
mutA
operon but does not affect its own promoter activity. The expressions of both
mutA
and
mutR
promoters are independent of the growth stage, while the production of mutacin II is only elevated at the early stationary phase. Taken together, these results suggest that expression of the mutacin operon is regulated by a complex system involving transcriptional and posttranscriptional or posttranslational controls.
Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic
Bacillus
sp. Strain K-1
Authors: Ratanakhanokchai Khanok , Kyu Khin Lay , Tanticharoen Morakot
Published: December 2019

An alkaliphilic bacterium,
Bacillus
sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe
2+
, Ca
2+
, and Mg
2+
greatly increased the xylanase activity, whereas Mn
2+
strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.
Estimating Bacterial Growth Parameters by Means of Detection Times
Authors: Baranyi József , Pin Carmen
Published: December 2019

We developed a new numerical method to estimate bacterial growth parameters by means of detection times generated by different initial counts. The observed detection times are subjected to a transformation involving the (unknown) maximum specific growth rate and the (known) ratios between the different inoculum sizes and the constant detectable level of counts. We present an analysis of variance (ANOVA) protocol based on a theoretical result according to which, if the specific rate used for the transformation is correct, the transformed values are scattered around the same mean irrespective of the original inoculum sizes. That mean, termed the physiological state of the inoculum,α̂, and the maximum specific growth rate, μ, can be estimated by minimizing the variance ratio of the ANOVA procedure. The lag time of the population can be calculated as λ = −ln α̂/μ; i.e. the lag is inversely proportional to the maximum specific growth rate and depends on the initial physiological state of the population. The more accurately the cell number at the detection level is known, the better the estimate for the variance of the lag times of the individual cells.
Lethal Effect ofRickettsia rickettsiion Its Tick Vector (Dermacentor andersoni)
Authors: Niebylski Mark L. , Peacock Mort G. , Schwan Tom G.
Published: December 2019

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88.3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4°C had a lower mortality (80.9%) than did those held at 21°C (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect ofR. rickettsiimay explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.
High-Level Formation of Active
Pseudomonas cepacia
Lipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in
Escherichia coli
and Rapid In Vitro Refolding
Authors: Quyen Dinh Thi , Schmidt-Dannert Claudia , Schmid Rolf D.
Published: December 2019

The lipase from
Pseudomonas cepacia
ATCC 21808 (recently reclassified as
Burkholderia cepacia
) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant
P. cepacia
harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in
Escherichia coli
were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λP
RL
promoter. However, no overexpression of the lipase chaperone was achieved in
E. coli
. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant
P. cepacia
per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in
E. coli
. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active
Pseudomonas
lipase (classes I and II) expressed in
E. coli
with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of
E. coli
wet cells.
Evaluation of Immunomagnetic Separation for Recovery of Infectious
Cryptosporidium parvum
Oocysts from Environmental Samples
Authors: Rochelle Paul A. , De Leon Ricardo , Johnson Anne , Stewart Mic H. , Wolfe Roy L.
Published: December 2019

Two commercial immunomagnetic separation (IMS) kits for
Cryptosporidium
were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity.
Biodiversity of
Lactococcus garvieae
Strains Isolated from Fish in Europe, Asia, and Australia
Authors: Eldar Avi , Goria Mariella , Ghittino Claudio , Zlotkin Amir , Bercovier Herve
Published: December 2019

Lactococcus garvieae
(junior synonym,
Enterococcus seriolicida
) is a major pathogen of fish, producing fatal septicemia among fish species living in very diverse environments. The phenotypic traits of
L. garvieae
strains collected from three different continents (Asia, Europe, and Australia) indicated phenotypic heterogeneity. On the basis of the acidification of
d
-tagatose and sucrose, three biotypes were defined. DNA relatedness values and a specific PCR assay showed that all the biotypes belonged to the same genospecies,
L. garvieae
. All of the
L. garvieae
strains were serotyped as Lancefield group N. Ribotyping proved that one clone was found both in Japan, where it probably originated, and in Italy, where it was probably imported. PCR of environmental samples did not reveal the source of the contamination of the fish in Italy. Specific clones (ribotypes) were found in outbreaks in Spain and in Italy. The
L. garvieae
reference strain, isolated in the United Kingdom from a cow, belonged to a unique ribotype.
L. garvieae
is a rising zoonotic agent. The biotyping scheme, the ribotyping analysis, and the PCR assay described in this work allowed the proper identification of
L. garvieae
and the description of the origin and of the source of contamination of strains involved in outbreaks or in sporadic cases.