bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Mitochondrial Toxin Produced by
Streptomyces griseus
Strains Isolated from an Indoor Environment Is Valinomycin
Authors: Andersson M. A. , Mikkola R. , Kroppenstedt R. M. , Rainey F. A. , Peltola J. , Helin J. , Sivonen K. , Salkinoja-Salonen M. S. 
Published: December 2019

ABSTRACT

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC
50
) were 10 to 63 ng (dry weight) ml of extended boar semen
−1
. The four strains were identified as
Streptomyces griseus
strains by 16S ribosomal DNA and chemotaxonomic methods. The four
S. griseus
strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml
−1
. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of
S. griseus
. The EC
50
of purified toxin from the
S. griseus
strains were 1 to 3 ng ml of extended boar semen
−1
, and the EC
50
of commercial valinomycin was 2 ng ml of extended boar semen
−1
. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as
S. griseus.
A Technique To Quantify the Population Size and Composition of the Biofilm Component in Communities of Bacteria in the Phyllosphere
Authors: Morris Cindy E. , Monier Jean-Michel , Jacques Marie-Agnès 
Published: December 2019

ABSTRACT
The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.
Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures
Authors: Master Emma R. , Mohn William W. 
Published: December 2019

ABSTRACT

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus
Pseudomonas
. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile
Burkholderia cepacia
LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.
Cloning of theAlcaligenes latusPolyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) inEscherichia coli
Authors: Choi Jong-il , Lee Sang Yup , Han Kyuboem 
Published: December 2019

ABSTRACTPolyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. RecombinantEscherichia colistrains harboring theRalstonia eutrophaPHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinantE. colistrains has been lower than that obtained with the wild-type bacteriumAlcaligenes latus. To endow the potentially superior PHA biosynthetic machinery toE. coli, we cloned the PHA biosynthesis genes fromA. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter inE. coli. RecombinantE. colistrains harboring theA. latusPHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring theR. eutrophagenes. With a pH-stat fed-batch culture of recombinantE. coliharboring a stable plasmid containing theA. latusPHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinantE. colito be used for the production of PHB with a high level of economic competitiveness.
NADP-Isocitrate Dehydrogenase from
Pseudomonas nautica
: Kinetic Constant Determination and Carbon Limitation Effects on the Pool of Intracellular Substrates
Authors: Roy Sylvie O. , Packard Ted T. 
Published: December 2019

ABSTRACT

Variations of intracellular concentrations of isocitrate and NADP
+
were measured throughout all growth phases of the marine bacterium
Pseudomonas nautica
. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP
+
and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP
+
concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP
+
concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP
+
, plays the larger role in the control of the respiratory CO
2
production rate (
R

CO
2

). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP
+
-specific isocitrate dehydrogenase (IDH) from
P. nautica
. These studies support the hypothesis that the mechanism of IDH’s activity involves the ordered addition of the substrates,
d
-isocitrate and NADP
+
. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to model
R

CO
2

in
P. nautica.
Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms
Authors: Großkopf Regine , Stubner Stephan , Liesack Werner 
Published: December 2019

ABSTRACTBecause excised, washed roots of rice (Oryza sativa) immediately produce CH4when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol. 21:25–36, 1996), we employed a culture-independent molecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were amplified directly from total root DNA by PCR and then cloned. Thirty-two archaeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca. 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanogens; 5 were affiliated withMethanosarcinaspp., and 5 were affiliated withMethanobacteriumspp. The remaining 22 ARR gene clones formed four distinct lineages (rice clusters I through IV) which were not closely related to any known cultured member of theArchaea. Rice clusters I and II formed distinct clades within the phylogenetic radiation of the orders “Methanosarcinales” andMethanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch asThermoplasma acidophilumand marine group II but was only distantly related to these taxa. Rice cluster IV was a deep-branching crenarchaeotal assemblage that was closely related to clone pGrfC26, an environmental sequence recovered from a temperate marsh environment. The use of a domain-specific oligonucleotide probe in a fluorescent in situ hybridization analysis revealed that viable members of theArchaeawere present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, designated rice cluster V, which was detected in anoxic rice paddy soil. These results indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.
An Immunological Strategy To Monitor In Situ the Phosphate Starvation State in
Thiobacillus ferrooxidans
Authors: Varela Patricia , Levicán Gloria , Rivera Francisco , Jerez Carlos A. 
Published: December 2019

ABSTRACT

Thiobacillus ferrooxidans
is one of the chemolithoautotrophic bacteria important in industrial biomining operations. During the process of ore bioleaching, the microorganisms are subjected to several stressing conditions, including the lack of some essential nutrients, which can affect the rates and yields of bioleaching. When
T. ferrooxidans
is starved for phosphate, the cells respond by inducing the synthesis of several proteins, some of which are outer membrane proteins of high molecular weight (70,000 to 80,000). These proteins were considered to be potential markers of the phosphate starvation state of these microorganisms. We developed a single-cell immunofluorescence assay that allowed monitoring of the phosphate starvation condition of this biomining microorganism by measuring the increased expression of the surface proteins. In the presence of low levels of arsenate (2 mM), the growth of phosphate-starved
T. ferrooxidans
cells was greatly inhibited compared to that of control nonstarved cells. Therefore, the determination of the phosphorus nutritional state is particularly relevant when arsenic compounds are solubilized during the bioleaching of different ores.
Recovery of Waterborne
Cryptosporidium parvum
Oocysts by Freshwater Benthic Clams (
Corbicula fluminea
)
Authors: Graczyk Thaddeus K. , Fayer Ronald , Cranfield Michael R. , Conn David Bruce 
Published: December 2019

ABSTRACT

Asian freshwater clams,
Corbicula fluminea
, exposed for 24 h to 38 liters of water contaminated with infectious
Cryptosporidium parvum
oocysts (1.00 × 10
6
oocysts/liter; approximately 1.9 × 10
5
oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne
C. parvum
oocysts. To optimize the detection of
C. parvum
oocysts in
C. fluminea
tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MER
IFLUOR
test kit).
Biphasic Thermal Inactivation Kinetics in
Salmonella enteritidis
PT4
Authors: Humpheson L. , Adams M. R. , Anderson W. A. , Cole M. B. 
Published: December 2019

ABSTRACT

The thermal inactivation kinetics of
Salmonella enteritidis
PT4 between 49 and 60°C were investigated. Using procedures designed to eliminate methodological artifacts, we found that the death kinetics deviated from the accepted model of first-order inactivation. When we used high-density stationary-phase populations and sensitive enumeration, the survivor curves at 60°C were reproducibly biphasic. The decimal reduction time at 60°C (
D
60°C
) of the tail subpopulation was more than four times that of the majority population. This difference decreased with decreasing temperature; i.e., the survivor curves became more linear, but the proportion of tail cells remained a constant proportion of the initial population, about 1 in 10
4
to 10
5
. Z plots (log
D
versus temperature) for the two populations showed that the
D
values coincided at 51°C, indicating that the survivor curves should be linear at this temperature, and this was confirmed experimentally. Investigations into the nature of the tails ruled out genotypic differences between the populations and protection due to leakage from early heat casualties. Heating of cells at 59°C in the presence of 5 or 100 μg of chloramphenicol per ml resulted in reductions in the levels of tailing. These reductions were greatest at the higher chloramphenicol concentration. Our results indicate that de novo protein synthesis of heat shock proteins is responsible for the observed tailing. Chemostat-cultured cells heated at 60°C also produced biphasic survivor curves in all but one instance. Cells with higher growth rates were more heat sensitive, but tailing was comparable with batch cultures. Starved cells (no dilution input) displayed linear inactivation kinetics, suggesting that during starvation a rapid heat shock response cannot be initiated.
Characterization of Chitinase C from a Marine Bacterium,
Alteromonas
sp. Strain O-7, and Its Corresponding Gene and Domain Structure
Authors: Tsujibo Hiroshi , Orikoshi Hideyuki , Shiotani Kayoko , Hayashi Miyuki , Umeda Junko , Miyamoto Katsushiro , Imada Chiaki , Okami Yoshiro , Inamori Yoshihiko 
Published: December 2019

ABSTRACT

One of the chitinase genes of
Alteromonas
sp. strain O-7, the chitinase C-encoding gene (
chiC
), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione
S
-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.
Effect of Selected Monoterpenes on Methane Oxidation, Denitrification, and Aerobic Metabolism by Bacteria in Pure Culture
Authors: Amaral J. A. , Ekins A. , Richards S. R. , Knowles R. 
Published: December 2019

ABSTRACTSelected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporiumOB3b,Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation byMethylosinus trichosporiumOB3b with 1.1 mM (−)-α-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was;Escherichia coli(optical density, 0.01), for example, was not affected by up to 7.3 mM (−)-α-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (−)-α-pinene, γ-terpinene, or β-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (−)-α-pinene, (S)-(−)-limonene, (R)-(+)-limonene, and γ-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, α-pinene oxide, linalool, α-terpineol] and a noncyclic hydrocarbon (β-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.
Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificity
l
-Threonine Aldolase from
Pseudomonas
sp. Strain NCIMB 10558
Authors: Liu Ji-Quan , Ito Saeko , Dairi Tohru , Itoh Nobuya , Kataoka Michihiko , Shimizu Sakayu , Yamada Hideaki 
Published: December 2019

ABSTRACT

A low-specificity
l
-threonine aldolase (
l
-TA) gene from
Pseudomonas
sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in
Escherichia coli
cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictly
l
specific at the α position, whereas it cannot distinguish between
threo
and
erythro
forms at the β position. In addition to threonine, the enzyme also acts on various other
l
-β-hydroxy-α-amino acids, including
l
-β-3,4-dihydroxyphenylserine,
l
-β-3,4-methylenedioxyphenylserine, and
l
-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity
l
-TA from
Saccharomyces cerevisiae
,
l
-
allo
-threonine aldolase from
Aeromonas jandaei
, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity
l
-TA from
Pseudomonas
sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the
l
-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.
Altered Specificity of Lactococcal Proteinase P
I
(Lactocepin I) in Humectant Systems Reflecting the Water Activity and Salt Content of Cheddar Cheese
Authors: Reid Julian R. , Coolbear Tim 
Published: December 2019

ABSTRACT

By using various humectant systems, the specificity of hydrolysis of α
s1
-, β-, and κ-caseins by the cell envelope-associated proteinase (lactocepin; EC
3.4.21.96
) with type P
1
specificity (i.e., lactocepin I) from
Lactococcus lactis
subsp.
lactis
BN1 was investigated at water activities (a
w
) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (a
w
= 0.95), hydrolysis of β-casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type P
III
enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from κ-casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (a
w
= 0.99). In addition, α
s1
-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an a
w
of 0.95) was generally as expected for lactocepin I activity except that β-casein peptide 47-52 and κ-casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of a
w
and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.