bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

Selected 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

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

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.
Induction of Mannanase, Xylanase, and Endoglucanase Activities inSclerotium rolfsii
Authors: Sachslehner Alois , Nidetzky Bernd , Kulbe Klaus D. , Haltrich Dietmar
Published: December 2019

Induction of mannanase, xylanase, and cellulase (endoglucanase) synthesis in the plant-pathogenic basidiomyceteSclerotium rolfsiiwas studied by incubating noninduced, resting mycelia with a number of mono-, oligo-, and polysaccharides. The simultaneous formation of these three endoglycanases could be provoked by several polysaccharides structurally resembling the carbohydrate constituents of lignocellulose (e.g., mannan and cellulose), by various disaccharide catabolites of these lignocellulose constituents (e.g., cellobiose, mannobiose, and xylobiose), or by structurally related disaccharides (e.g., lactose, sophorose, and galactosyl-β-1,4-mannose), as well as byl-sorbose. Synthesis of mannanase, xylanase, and endoglucanase always occurred concomitantly and could not be separated by selecting an appropriate inducer. Various structurally different inducing carbohydrates promoted the excretion of the same multiple isoforms of endoglycanases, as judged from the similar banding patterns obtained in zymogram analyses of enzyme preparations obtained in response to these different inducers and resolved by analytical isoelectric focusing. Whereas enhanced xylanase and endoglucanase formation is strictly dependent on the presence of suitable inducers, increased levels of mannanase are excreted byS. rolfsiieven under noninducing, derepressed conditions, as shown in growth experiments with glucose as the substrate. Significant mannanase formation commenced only when glucose was exhausted from the medium. Under these conditions, only very low, presumably constitutive levels of xylanase and endoglucanase were formed. Although the induction of the three endoglycanases is very closely related inS. rolfsii, it was concluded that there is no common, coordinated regulatory mechanism that controls the synthesis of mannanase, xylanase, and endoglucanase.
Metabolism of Dichloromethane by the Strict Anaerobe
Dehalobacterium formicoaceticum
Authors: Mägli Andreas , Messmer Michael , Leisinger Thomas
Published: December 2019

The metabolism of dichloromethane by
Dehalobacterium formicoaceticum
in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [
13
C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO
2
from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 μM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane.
D. formicoaceticum
exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited by
N
,
N
′-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F
0
F
1
-type ATP synthase.
Extended Survival and Persistence of
Campylobacter
spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining
Authors: Buswell Clive M. , Herlihy Yvonne M. , Lawrence Lorna M. , McGuiggan James T. M. , Marsh Philip D. , Keevil C. William , Leach Steve A.
Published: December 2019

In water microcosm experiments, the survival times of
Campylobacter
isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora,
Campylobacter
isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a
Campylobacter
-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.
Engineering of Quasi-Natural
Pseudomonas putida
Strains for Toluene Metabolism through an
ortho
-Cleavage Degradation Pathway
Authors: Panke Sven , Sánchez-Romero Juan M. , de Lorenzo Víctor
Published: December 2019

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of
Pseudomonas putida
was fully reassembled as a single gene cassette along with its cognate regulatory gene,
xylR
. The corresponding DNA segment was then targeted to the chromosome of a
P. putida
strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping
ortho
-ring cleavage pathway of the catechol intermediate.
Environmental Occurrence of the Whipple’s Disease Bacterium (
Tropheryma whippelii
)
Authors: Maiwald Matthias , Schuhmacher Frank , Ditton Hans-Jürgen , von Herbay Axel
Published: December 2019

Whipple’s disease is a systemic disorder in which a gram-positive rod-shaped bacterium is constantly present in infected tissues. After numerous unsuccessful attempts to culture this bacterium, it was eventually characterized by 16S rRNA gene analysis to be a member of the actinomycetes. The name
Tropheryma whippelii
was proposed. Until now, the bacterium has only been found in infected human tissues, but there is no evidence for human-to-human transmission. Here we report the detection of DNA specific for the Whipple’s disease bacterium in 25 of 38 wastewater samples from five different sewage treatment plants in the area of Heidelberg, Germany. These findings provide the first evidence that
T. whippelii
occurs in the environment, within a polymicrobial community. This is in accordance with the phylogenetic relationship of this bacterium as well as with known epidemiological aspects of Whipple’s disease. Our data argue for an environmental source for infection with the Whipple’s disease bacterium.
An Anaerobic, Intrachamber Incubator for Growth of
Methanosarcina
spp. on Methanol-Containing Solid Media
Authors: Metcalf William W. , Zhang Jun Kai , Wolfe Ralph S.
Published: December 2019

To simplify the incubation of
Methanosarcina
spp. on solid agar medium, a two-port, manual, rectangular air lock was modified to serve as an anaerobic incubator. In one operation, it is possible to incubate 153 petri plates, the equivalent of 11 standard anaerobic jars, with plating efficiencies identical to those of traditional protocols.
Inactivation of
Cryptosporidium parvum
Oocysts by Ammonia
Authors: Jenkins Michael B. , Bowman Dwight D. , Ghiorse William C.
Published: December 2019

The survival of
Cryptosporidium parvum
oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH
3
) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h
−1
. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.
Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA
Authors: Marchesi Julian R. , Sato Takuichi , Weightman Andrew J. , Martin Tracey A. , Fry John C. , Hiom Sarah J. , Wade William G.
Published: December 2019

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.
Purification, Characterization, and Mechanism of a Flavin Mononucleotide-Dependent 2-Nitropropane Dioxygenase from
Neurospora crassa
Authors: Gorlatova Natalia , Tchorzewski Marek , Kurihara Tatsuo , Soda Kenji , Esaki Nobuyoshi
Published: December 2019

A nitroalkane-oxidizing enzyme was purified to homogeneity from
Neurospora crassa
. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an
18
O
2
atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH
3
CH(NO
2
)-CH
3
+ O
2
→2CH
3
COCH
3
+ 2HNO
2
, which is identical to that of the reaction of 2-nitropropane dioxygenase from
Hansenula mrakii
. The reaction of the
Neurospora
enzyme was inhibited by superoxide anion scavengers in the same manner as that of the
Hansenula
enzyme. Both of these enzymes are flavoenzymes; however, the
Neurospora
enzyme contains flavin mononucleotide as a prosthetic group, whereas the
Hansenula
enzyme contains flavin adenine dinucleotide.
Microbial Reductive Dechlorination of Aroclor 1260 in Anaerobic Slurries of Estuarine Sediments
Authors: Wu Qingzhong , Sowers Kevin R. , May Harold D.
Published: December 2019

Reductive dechlorination of Aroclor 1260 was investigated in anaerobic slurries of estuarine sediments from Baltimore Harbor (Baltimore, Md.). The sediment slurries were amended with 800 ppm Aroclor 1260 with and without the addition of 350 μM 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) or 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) and incubated in triplicate at 30°C under methanogenic conditions in an artificial estuarine medium. After 6 months, extensive
meta
dechlorination and moderate
ortho
dechlorination of Aroclor 1260 occurred in all incubated cultures except for sterilized controls. Overall, total chlorines per biphenyl decreased by up to 34%.
meta
chlorines per biphenyl decreased by 65, 55, and 45% and
ortho
chlorines declined by 18, 12, and 9%, respectively, when 2,3,4,5-CB, 2,3,5,6-CB, or no additional congener was supplied. This is the first confirmed report of microbial
ortho
dechlorination of a commercial polychlorinated biphenyl mixture. In addition, compared with incubated cultures supplied with Aroclor 1260 alone, the dechlorination of Aroclor 1260 plus 2,3,4,5-CB or 2,3,5,6-CB occurred with shorter lag times (31 to 60 days versus 90 days) and was more extensive, indicating that the addition of a single congener stimulated the dechlorination of Aroclor 1260.
Expression of
Clostridium acetobutylicum
ATCC 824 Genes in
Escherichia coli
for Acetone Production and Acetate Detoxification
Authors: Bermejo Lourdes L. , Welker Neil E. , Papoutsakis Eleftherios T.
Published: December 2019

A synthetic acetone operon (
ace4
) composed of four
Clostridium acetobutylicum
ATCC 824 genes (
adc
,
ctfAB
, and
thl
, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the
thl
promoter was constructed and was introduced into
Escherichia coli
on vector pACT. Acetone production demonstrated that
ace4
is expressed in
E. coli
and resulted in the reduction of acetic acid levels in the fermentation broth. Since different
E. coli
strains vary significantly in their growth characteristics and acetate metabolism,
ace4
was expressed in three
E. coli
strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type
C. acetobutylicum
. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing
E. coli
strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided.
Characterization of EngF from
Clostridium cellulovorans
and Identification of a Novel Cellulose Binding Domain
Authors: Ichi-ishi Akihiko , Sheweita Salah , Doi Roy H.
Published: December 2019

The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from
Clostridium cellulovorans
were studied. Binding studies revealed that the

K
d

and the maximum amount of protein bound for acid-swollen cellulose were 1.8 μM and 7.1 μmol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.
Surface Charge Properties of and Cu(II) Adsorption by Spores of the Marine
Bacillus
sp. Strain SG-1
Authors: He Lee M. , Tebo Bradley M.
Published: December 2019

Spores of marine
Bacillus
sp. strain SG-1 are capable of oxidizing Mn(II) and Co(II), which results in the precipitation of Mn(III, IV) and Co(III) oxides and hydroxides on the spore surface. The spores also bind other heavy metals; however, little is known about the mechanism and capacity of this metal binding. In this study the characteristics of the spore surface and Cu(II) adsorption to this surface were investigated. The specific surface area of wet SG-1 spores was 74.7 m
2
per g of dry weight as measured by the methylene blue adsorption method. This surface area is 11-fold greater than the surface area of dried spores, as determined with an N
2
adsorption surface area analyzer or as calculated from the spore dimensions, suggesting that the spore surface is porous. The surface exchange capacity as measured by the proton exchange method was found to be 30.6 μmol m
−2
, which is equal to a surface site density of 18.3 sites nm
−2
. The SG-1 spore surface charge characteristics were obtained from acid-base titration data. The surface charge density varied with pH, and the zero point of charge was pH 4.5. The titration curves suggest that the spore surface is dominated by negatively charged sites that are largely carboxylate groups but also phosphate groups. Copper adsorption by SG-1 spores was rapid and complete within minutes. The spores exhibited a high affinity for Cu(II). The amounts of copper adsorbed increased from negligible at pH 3 to maximum levels at pH >6. Their great surface area, site density, and affinity give SG-1 spores a high capability for binding metals on their surfaces, as demonstrated by our experiments with Cu(II).
Common Elements Regulating Gene Expression in Temperate and Lytic Bacteriophages of
Lactococcus
Species
Authors: Walker Shirley A. , Dombroski Carol S. , Klaenhammer Todd R.
Published: December 2019

A phage-inducible middle promoter (P
15A10
) from the lytic, lactococcal bacteriophage φ31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, φr1t and φLC3. Sequencing upstream and downstream of P
15A10
showed that the high degree of homology between φ31 and φr1t continued beyond the phage promoter. With the exception of one extra open reading frame in φ31, the sequences were highly homologous (95 to 98%) between nucleotides 13448 and 16320 of the published φr1t sequence. By use of a β-galactosidase (β-Gal) gene under the control of a smaller, more tightly regulated region within the P
15A10
promoter, P
566–888
, it was established that mitomycin C induction of a lactococcal strain harboring the prophage φr1t induced the P
566–888
promoter, as determined from an increase in β-Gal activity. Hybridization of nine other lactococcal strains with
32
P-labeled P
566–888
showed that the
Lactococcus lactis
strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P
566–888
promoter, as determined from an increase in β-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of φr1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P
566–888
from the lytic phage φ31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage φ31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.
Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval
Authors: Großkopf Regine , Janssen Peter H. , Liesack Werner
Published: December 2019

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with
Methanobacterium bryantii
and
Methanobacterium formicicum
, six were closely related but not identical to those of strains of
Methanosaeta concilii
, two grouped with members of the genus
Methanosarcina
, and two were related to the methanogenic endosymbiont of
Plagiopyla nasuta
. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10
7
per g of dry soil for the H
2
-CO
2
-utilizing methanogens and of up to 10
6
for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H
2
-CO
2
, grouped within the phylogenetic radiation characterized by
M. bryantii
and
M. formicicum
and the environmental sequences of the
Methanobacterium
-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel
Methanosaeta
-like group and the
Methanobacterium
-like group.
Methanosarcina
spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known
Methanosarcina
spp. but are at the acetate utilization threshold of
Methanosaeta
spp. Thus, our data indicated
Methanosaeta
spp. and
Methanobacterium
spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas
Methanosarcina
spp. appeared to be less abundant.
Relationship between Intracellular Phosphate, Proton Motive Force, and Rate of Nongrowth Energy Dissipation (Energy Spilling) in
Streptococcus bovis
JB1
Authors: Bond Daniel R. , Russell James B.
Published: December 2019

When the rate of glucose addition to nongrowing
Streptococcus bovis
cell suspensions was increased, the fermentation was homolactic, fructose-1,6-diphosphate (FDP) increased, intracellular inorganic phosphate (P
i
) declined, and the energy-spilling rate increased. ATP and ADP were not significantly affected by glucose consumption rate, but the decrease in P
i
was sufficient to cause an increase in the free energy of ATP hydrolysis (ΔG′p). The increase in ΔG′p was correlated with an increase in proton motive force (Δp).
S. bovis
continuous cultures (dilution rate of 0.65 h
−1
) that were provided with ammonia as the sole nitrogen source also had high rates of lactate production and energy spilling. When Trypticase was added as a source of amino acids, lactate production decreased; a greater fraction of the glucose was converted to acetate, formate, and ethanol; and the energy-spilling rate decreased. Trypticase also caused a decrease in FDP, an increase in P
i
, and a decrease in Δp. The change in Δp could be explained by P
i
-dependent changes in the ΔG′p. When P
i
declined, ΔG′p and Δp increased. The ratio of ΔG′p to Δp (millivolt per millivolt) was always high (>4) at low rates of energy spilling but declined when the energy-spilling rate increased. Based on these results, it appears that Δp and the energy-spilling rate are responsive to fluctuations in the intracellular P
i
concentration.
Thermal Tolerance ofMycobacterium paratuberculosis
Authors: Sung Nackmoon , Collins Michael T.
Published: December 2019

Dvalues (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) ofMycobacterium paratuberculosisin 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71°C). ViableM. paratuberculosisorganisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for theM. paratuberculosisstrains tested were generally linear, withR2of ≥0.90, but a few curves (R2, 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similarDvalues in milk and in lactate solution. However,Dvalues for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum.Dvalues for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). TheDvalue based on pooled data for clinical strains ofM. paratuberculosisin milk at 71°C (D71°C) was 11.67 s. PooledD62°C,D65°C, andD68°Cof clinicalM. paratuberculosisstrains in milk were 228.8, 47.8, and 21.8 s, respectively. TheZvalue (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11°C. TheDvalues of clumped and singleM. paratuberculosiscells were not significantly different. TheDvalues of allM. paratuberculosisstrains tested were considerably higher than those published forListeria,Salmonella, andCoxiellaspp. and estimated forMycobacterium bovis, indicating thatM. paratuberculosisis more thermally tolerant. This study supports the premise thatM. paratuberculosismay survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 101cells/ml.
A Novel Sensitive Bioassay for Detection of
Bacillus cereus
Emetic Toxin and Related Depsipeptide Ionophores
Authors: Andersson M. A. , Mikkola R. , Helin J. , Andersson M. C. , Salkinoja-Salonen M.
Published: December 2019

Of the toxins produced by
Bacillus cereus
, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of
B. cereus
is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic
B. cereus
strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen
−1
. This amount corresponds to 10
4
to 10
5
CFU of
B. cereus
cells. No toxicity was detected for 27 other
B. cereus
strains up to 10
8
CFU ml
−1
. The detection limit for food was 3 g of rice containing 10
6
to 10
7
CFU of emetic
B. cereus
per gram. Effects similar to those provoked by emetic
B. cereus
toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen
−1
, respectively. The symptoms provoked by the toxin in spermatozoa indicated that
B. cereus
emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.
Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by
Nitrosomonas europaea
Authors: Hommes Norman G. , Russell Sterling A. , Bottomley Peter J. , Arp Daniel J.
Published: December 2019

Ammonia monooxygenase (AMO) from
Nitrosomonas europaea
catalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of
N. europaea
. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with
N. europaea
cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by
N. europaea
. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH
4
+
in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH
3
to concentrations (8 to 80 μM) similar to the

K
s

value of AMO for NH
3
(∼29 μM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.
Molecular Cloning and Transcriptional Regulation of theAspergillus nidulans xlnDGene Encoding a β-Xylosidase
Authors: Pérez-González José A. , van Peij Noël N. M. E. , Bezoen Alja , Maccabe Andrew P. , Ramón Daniel , de Graaff Leo H.
Published: December 2019

ThexlnDgene encoding the 85-kDa β-xylosidase was cloned fromAspergillus nidulans. The deduced primary structure of the protein exhibits considerable similarity to the primary structures of theAspergillus nigerandTrichoderma reeseiβ-xylosidases and some similarity to the primary structures of the class 3 β-glucosidases.xlnDis regulated at the transcriptional level; it is induced by xylan andd-xylose and is repressed byd-glucose. Glucose repression is mediated by the product of thecreAgene. Although several binding sites for the pH regulatory protein PacC were found in the upstream regulatory region, it was not clear from a Northern analysis whether PacC is involved in transcriptional regulation ofxlnD.
Analysis of the β-1,3-Glucanolytic System of the Biocontrol AgentTrichoderma harzianum
Authors: Vázquez-Garcidueñas Soledad , Leal-Morales Carlos A. , Herrera-Estrella Alfredo
Published: December 2019

The biocontrol agentTrichoderma harzianumIMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.
Use of
13
C Nuclear Magnetic Resonance To Assess Fossil Fuel Biodegradation: Fate of [1-
13
C]Acenaphthene in Creosote Polycyclic Aromatic Compound Mixtures Degraded by Bacteria
Authors: Selifonov Sergey A. , Chapman Peter J. , Akkerman Simon B. , Gurst Jerome E. , Bortiatynski Jacqueline M. , Nanny Mark A. , Hatcher Patrick G.
Published: December 2019

[1-
13
C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled
13
C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows
13
C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-
13
C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From
13
C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain
Pseudomonas
sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.