bluuebunny/crossref_metadata_2025 huggingface dataset:
Mannan-Degrading Enzymes from
Cellulomonas fimi
Authors: Stoll Dominik , Stålbrand Henrik , Warren R. Antony J.
Published: December 2019

The genes
man26a
and
man2A
from
Cellulomonas fimi
encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by
Escherichia coli
to
C. fimi
protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by
C. fimi
during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by
C. fimi
remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.
PCR Comparison of
Mycobacterium avium
Isolates Obtained from Patients and Foods
Authors: Yoder S. , Argueta C. , Holtzman A. , Aronson T. , Berlin O. G. W. , Tomasek P. , Glover N. , Froman S. , Stelma G.
Published: December 2019

Mycobacterium avium
is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS
1245
and IS
1311
to compare levels of relatedness of
M. avium
isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were
M. avium
. Twelve food and 103 clinical
M. avium
isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of
M. avium
infection. This study demonstrates a rapid, inexpensive method for typing
M. avium
, possibly replacing pulsed-field gel electrophoresis.
BACTOX, a Rapid Bioassay That Uses Protozoa To Assess the Toxicity of Bacteria
Authors: Schlimme Wolfram , Marchiani Marcello , Hanselmann Kurt , Jenni Bernard
Published: December 2019

A new type of toxicity test based on the protozoan
Tetrahymena pyriformis
has been developed to assess the overall toxicity of bacterial strains given as prey. This simple and rapid test is able to detect toxicant-producing bacteria, which may present a biohazard. It can also be used for the risk assessment of microbes designed for deliberate release.
Conjugal Transfer but Not Quorum-Dependent
tra
Gene Induction of pTiC58 Requires a Solid Surface
Authors: Piper Kevin R. , Farrand Stephen K.
Published: December 2019

Donors of
Agrobacterium tumefaciens
harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a
lacZ
reporter fusion, the
tra
genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate
tra
gene expression to equivalent levels in liquid and on solid surfaces.
Method for Detection and Enumeration ofCryptosporidium parvumOocysts in Feces, Manures, and Soils
Authors: Kuczynska Ewa , Shelton Daniel R.
Published: December 2019

Eight concentration and purification methods were evaluated to determine percentages of recovery ofCryptosporidium parvumoocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105oocysts g of bovine feces−1. The percentages of recovery ranged from 10.8% (25 oocysts g−1) to 17.0% (104oocysts g−1) (r2= 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces−1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (104oocysts g−1). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil−1depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.
Influence of Kernel Age on Fumonisin B
1
Production in Maize by
Fusarium moniliforme
Authors: Warfield Colleen Y. , Gilchrist David G.
Published: December 2019

Production of fumonisins by
Fusarium moniliforme
on naturally infected maize ears is an important food safety concern due to the toxic nature of this class of mycotoxins. Assessing the potential risk of fumonisin production in developing maize ears prior to harvest requires an understanding of the regulation of toxin biosynthesis during kernel maturation. We investigated the developmental-stage-dependent relationship between maize kernels and fumonisin B
1
production by using kernels collected at the blister (R2), milk (R3), dough (R4), and dent (R5) stages following inoculation in culture at their respective field moisture contents with
F. moniliforme
. Highly significant differences (
P
≤ 0.001) in fumonisin B
1
production were found among kernels at the different developmental stages. The highest levels of fumonisin B
1
were produced on the dent stage kernels, and the lowest levels were produced on the blister stage kernels. The differences in fumonisin B
1
production among kernels at the different developmental stages remained significant (
P
≤ 0.001) when the moisture contents of the kernels were adjusted to the same level prior to inoculation. We concluded that toxin production is affected by substrate composition as well as by moisture content. Our study also demonstrated that fumonisin B
1
biosynthesis on maize kernels is influenced by factors which vary with the developmental age of the tissue. The risk of fumonisin contamination may begin early in maize ear development and increases as the kernels reach physiological maturity.
Purification and Characterization of an α-Glucosidase from
Rhizobium
sp. (
Robinia pseudoacacia
L.) Strain USDA 4280
Authors: Berthelot Karine , Delmotte Francis M.
Published: December 2019

A novel α-glucosidase with an apparent subunit mass of 59 ± 0.5 kDa was purified from protein extracts of
Rhizobium
sp. strain USDA 4280, a nodulating strain of black locust (
Robinia pseudoacacia
L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 ± 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35°C. The activity increased in the presence of NH
4
+
and K
+
ions but was inhibited by Cu
2+
, Ag
+
, Hg
+
, and Fe
2+
ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl α-glucoside was the fluorogenic substrate. The enzyme was more active with α-glucosides substituted with aromatic aglycones than with oligosaccharides. This α-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl α-
d
-glucopyranoside (

K
m

, 0.141 μM;
V
max
, 6.79 μmol min
−1
mg
−1
) and with
p
-nitrophenyl α-
d
-glucopyranoside (

K
m

, 0.037 μM;
V
max
, 2.92 μmol min
−1
mg
−1
). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.
Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica)
Authors: Genthner Fred J. , Volety Aswani K. , Oliver Leah M. , Fisher William S.
Published: December 2019

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer.Vibrio parahaemolyticusstrains, altered in motility or colonial morphology (opaque and translucent), andListeria monocytogenesmutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer.Vibrio vulnificusstrains, opaque and translucent (with and without capsules), were examined only in summer. AmongV. parahaemolyticusandL. monocytogenes, significantly (P< 0.05) higher levels of killing by hemocytes were observed in summer than in winter.L. monocytogeneswas more resistant thanV. parahaemolyticusorV. vulnificusto the bactericidal activity of hemocytes. In winter, both translucent strains ofV. parahaemolyticusshowed significantly (P< 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of theV. parahaemolyticustranslucent strains showed significantly (P< 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P> 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs ofV. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabledV. parahaemolyticusto evade killing by the oyster’s cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.
A Sensitive Nested Reverse Transcriptase PCR Assay To Detect Viable Cells of the Fish Pathogen
Renibacterium salmoninarum
in Atlantic Salmon (
Salmo salar
L.)
Authors: Cook Marcia , Lynch William H.
Published: December 2019

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen
Renibacterium salmoninarum
in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (
Salmo salar
L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of
R. salmoninarum
. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable
R. salmoninarum
within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.
Purification and Characterization of a Secreted Laccase of
Gaeumannomyces graminis
var.
tritici
Authors: Edens William A. , Goins Tresa Q. , Dooley David , Henson Joan M.
Published: December 2019

We purified a secreted fungal laccase from filtrates of
Gaeumannomyces graminis
var.
tritici
cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphenol (

K
m

= 2.6 × 10
−5
± 7 × 10
−6
M), catechol (

K
m

= 2.5 × 10
−4
± 1 × 10
−5
M), pyrogallol (

K
m

= 3.1 × 10
−4
± 4 × 10
−5
M), and guaiacol (

K
m

= 5.1 × 10
−4
± 2 × 10
−5
M). In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen’s hyphae and/or in lignin depolymerization in its infected plant host.
Effect of Growth Temperature on Hydrolytic and Esterifying Activities from
Pseudomonas fragi
CRDA 037 Grown on Whey
Authors: Fonchy E. , Morin A. , Rodrigue N. , Müller B. , Chalier P.
Published: December 2019

The production of hydrolytic and esterifying activities of
Pseudomonas fragi
CRDA 037 grown on a whey-based medium was investigated at different temperatures over time. The optimal temperature was found to be critical and different for the production of both activities. The highest hydrolytic activity was detected with bacteria cultivated at between 24°C (149.2 U/liter) and 27°C (133.8 U/liter), while the highest production of ethyl valerate (esterifying activity) was observed by using biomass grown at 15°C (0.75 U/liter). When the fermentation temperature was increased, the incubation time necessary to reach the maximal concentration of both activities was reduced. Studies of the thermostability of both activities showed that the hydrolytic activity was more stable than the esterifying activity at 15 and 24°C. Statistical analysis allowed the determination of the equations that predicted the production of hydrolytic and esterifying activities as a function of time and growth temperature. The optimal assay temperatures for the hydrolytic and esterifying activities were 37°C and 12 to 15°C, respectively.
Identification of a New Gene Family Expressed during the Onset of Sexual Reproduction in the Centric Diatom
Thalassiosira weissflogii
Authors: Armbrust E. Virginia
Published: December 2019

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom
Thalassiosira weissflogii
initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes,
Sig1
,
Sig2
, and
Sig3
, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.
Discriminant Analysis of Ribotype Profiles of
Escherichia coli
for Differentiating Human and Nonhuman Sources of Fecal Pollution
Authors: Parveen Salina , Portier Kenneth M. , Robinson Kevin , Edmiston Lee , Tamplin Mark L.
Published: December 2019

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife.
Escherichia coli
is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238
E. coli
isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.
Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of
Lactobacillus fermentum
in Ghanaian Fermented Maize Dough
Authors: Hayford Alice E. , Petersen Anne , Vogensen Finn K. , Jakobsen Mogens
Published: December 2019

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant
Lactobacillus
isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included
Lactobacillus fermentum
, whereas cluster 2 comprised the remaining
Lactobacillus
spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of
L. fermentum
included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception,
Lactobacillus reuteri
LMG 13045, the probes hybridized only to fragments of different sizes in
Eco
RI-digested chromosomal DNA of
L. fermentum
strains, thus indicating the specificity of the probes and variation within the
L. fermentum
isolates.
Engineering of Stable Recombinant Bacteria for Production of Chiral Medium-Chain-Length Poly-3-Hydroxyalkanoates
Authors: Prieto Maria A. , Kellerhals Michele B. , Bozzato Gian B. , Radnovic Dragan , Witholt Bernard , Kessler Birgit
Published: December 2019

In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for
phaC1
, which encodes one of the mcl-PHA polymerases of
Pseudomonas oleovorans
. We used the mini-Tn
5
system as a tool to construct
Escherichia coli
193MC1 and
P. oleovorans
POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level.
E. coli
193MC1 produced considerably shorter polyesters than
P. oleovorans
produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that
E. coli
193MC1 incorporated exclusively the
R
configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.
Sulfidogenesis from 2-Aminoethanesulfonate (Taurine) Fermentation by a Morphologically Unusual Sulfate-Reducing Bacterium,Desulforhopalus singaporensissp. nov
Authors: Lie Thomas J. , Clawson Michael L. , Godchaux Walter , Leadbetter Edward R.
Published: December 2019

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The nameDesulforhopalus singaporensisis proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-β-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genusDesulforhopaluswhich has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297–301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation.D. singaporensisthus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.
Identification of the
yqhE
and
yafB
Genes Encoding Two 2,5-Diketo-
d
-Gluconate Reductases in
Escherichia coli
Authors: Yum Do-Young , Lee Bong-Yong , Pan Jae-Gu
Published: December 2019

The identification of a gene (
yiaE
) encoding 2-ketoaldonate reductase (2KR) in our previous work led to the hypothesis that
Escherichia coli
has other ketogluconate reductases including 2,5-diketo-
d
-gluconate reductase (25DKGR) and to study of the related ketogluconate metabolism. By using the deduced amino acid sequences of 5-diketo-
d
-gluconate reductase (5KDGR) of
Gluconobacter oxydans
and 25DKGR of
Corynebacterium
sp., protein databases were screened to detect homologous proteins. Among the proteins of
E. coli
, an oxidoreductase encoded by
yjgU
and having 56% similarity to 5KDGR of
G. oxydans
and two hypothetical oxidoreductases encoded by
yqhE
and
yafB
and having 49.8 and 42% similarity, respectively, to 25DKGR of
Corynebacterium
sp. were detected. Recently, the
yjgU
gene was identified as encoding 5KDGR and renamed
idnO
(C. Bausch, N. Peekhaus, C. Utz, T. Blais, E. Murray, T. Lowary, and T. Conway, J. Bacteriol. 180:3704–3710, 1998). The pathways involved in the metabolism of ketogluconate by
E. coli
have been predicted by biochemical analysis of purified enzymes and chemical analysis of the pathway intermediates. The gene products of
yqhE
and
yafB
were identified as 25DKGR-A, and 25DKGR-B, respectively, catalyzing the reduction of 25KDG to 2-keto-
l
-gulonate (2KLG). The native 25DKGR-A, 25DKGR-B, and 5KDGR had apparent molecular weights of about 30,000, 30,000, and 54,000, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, all three enzymes showed protein bands with a molecular weight of about 29,000, which indicated that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, and dimeric proteins, respectively. The optimum pHs for reduction were 7.5, 7.0, and 8.0, respectively. The 5KDGR was active with NADH, whereas 25DKGR-A and 25DKGR-B were active with NADPH as a preferred electron donor. 25DKG can be converted to 5KDG by 2KR, which is then reduced to
d
-gluconate by 5KDGR. The pathways were compared with those of
Erwinia
sp. and
Corynebacterium
sp. A BLAST search of published and incomplete microbial genome sequences revealed that the ketogluconate reductases and their related metabolism may be widespread in many species.
Factors Influencing Expression ofluxCDABEandnahGenes inPseudomonas putidaRB1353(NAH7, pUTK9) in Dynamic Systems
Authors: Neilson Julia W. , Pierce Shelley A. , Maier Raina M.
Published: December 2019

Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading,luxbioreporter bacteriumPseudomonas putidaRB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter−1. Oxygen tension was evaluated over the range of 0.5 to 40 mg liter−1, with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter−1) observed at 1.5 mg liter−1and below and no effect observed above 5 mg liter−1. Oxygen tensions from 2 to 4 mg liter−1influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of thenahoperon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates thatluxreporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, eachluxreporter system and application will require characterization and calibration.
Microbial Population Changes during Bioremediation of an Experimental Oil Spill
Authors: MacNaughton Sarah J. , Stephen John R. , Venosa Albert D. , Davis Gregory A. , Chang Yun-Juan , White David C.
Published: December 2019

Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the α-proteobacteria and
Flexibacter-Cytophaga-Bacteroides
phylum. α-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.
Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil
Authors: Nüsslein Klaus , Tiedje James M.
Published: December 2019

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from
Fibrobacter
and
Syntrophomonas
assemblages in the forest soil to
Burkholderia
and
Rhizobium–Agrobacterium
assemblages in the pasture soil.
Sporogenic Effect of Polyunsaturated Fatty Acids on Development of
Aspergillus
spp
Authors: Calvo Ana M. , Hinze Lori L. , Gardner Harold W. , Keller Nancy P.
Published: December 2019

Aspergillus
spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in
Aspergillus nidulans
,
Aspergillus flavus
, and
Aspergillus parasiticus
is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in
A. flavus
and
A. parasiticus
strains and altered sclerotium production in some
A. flavus
strains in which sclerotium production decreases in the light but increases in the dark. In
A. nidulans
, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on
A. nidulans
are similar to the sporogenic effects of
A. nidulans
psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on
A. nidulans
required an intact
veA
gene. The sporogenic effects of light and linoleic acid on
Aspergillus
spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.
Transformation of Sulfur Compounds by an Abundant Lineage of Marine Bacteria in the α-Subclass of the Class
Proteobacteria
Authors: González José M. , Kiene Ronald P. , Moran Mary Ann
Published: December 2019

Members of a group of marine bacteria that is numerically important in coastal seawater and sediments were characterized with respect to their ability to transform organic and inorganic sulfur compounds. Fifteen strains representing the
Roseobacter
group (a phylogenetic cluster of marine bacteria in the α-subclass of the class
Proteobacteria
) were isolated from seawater, primarily from the southeastern United States. Although more than one-half of the isolates were obtained without any selection for sulfur metabolism, all of the isolates were able to degrade the sulfur-containing osmolyte dimethyl sulfoniopropionate (DMSP) with production of dimethyl sulfide (DMS). Five isolates also degraded DMSP with production of methanethiol, indicating that both cleavage and demethylation pathways for DMSP occurred in the same organism, which is unusual. Five isolates were able to reduce dimethyl sulfoxide to DMS, and several isolates also degraded DMS and methanethiol. Sulfite oxygenase activity and methanesulfonic acid oxygenase activity were also present in some of the isolates. The ability to incorporate the reduced sulfur in DMSP and methanethiol into cellular material was studied with one of the isolates. A group-specific 16S rRNA probe indicated that the relative abundance of uncultured bacteria in the
Roseobacter
group increased in seawater enriched with DMSP or DMS. Because this group typically accounts for >10% of the 16S ribosomal DNA pool in coastal seawater and sediments of the southern United States, clues about its potential biogeochemical role are of particular interest. Studies of culturable representatives suggested that the group could mediate a number of steps in the cycling of both organic and inorganic forms of sulfur in marine environments.
Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil
Authors: Deni Jamal , Penninckx Michel J.
Published: December 2019

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.
Development and Field Performance of a Broad-Spectrum Nonviable Asporogenic Recombinant Strain of
Bacillus thuringiensis
with Greater Potency and UV Resistance
Authors: Sanchis Vincent , Gohar Michel , Chaufaux Josette , Arantes Olivia , Meier Alain , Agaisse Hervé , Cayley Jane , Lereclus Didier
Published: December 2019

The main problems with
Bacillus thuringiensis
products for pest control are their often narrow activity spectrum, high sensitivity to UV degradation, and low cost effectiveness (high potency required). We constructed a sporulation-deficient SigK
−
B. thuringiensis
strain that expressed a chimeric
cry1C/Ab
gene, the product of which had high activity against various lepidopteran pests, including
Spodoptera littoralis
(Egyptian cotton leaf worm) and
Spodoptera exigua
(lesser [beet] armyworm), which are not readily controlled by other Cry δ-endotoxins. The SigK
−
host strain carried the
cry1Ac
gene, the product of which is highly active against the larvae of the major pests
Ostrinia nubilalis
(European corn borer) and
Heliothis virescens
(tobacco budworm). This new strain had greater potency and a broader activity spectrum than the parent strain. The crystals produced by the asporogenic strain remained encapsulated within the cells, which protected them from UV degradation. The
cry1C/Ab
gene was introduced into the
B. thuringiensis
host via a site-specific recombination vector so that unwanted DNA was eliminated. Therefore, the final construct contained no sequences of non-
B. thuringiensis
origin. As the recombinant strain is a mutant blocked at late sporulation, it does not produce viable spores and therefore cannot compete with wild-type
B. thuringiensis
strains in the environment. It is thus a very safe biopesticide. In field trials, this new recombinant strain protected cabbage and broccoli against a pest complex under natural infestation conditions.
Identification of an ATP-Driven, Osmoregulated Glycine Betaine Transport System in
Listeria monocytogenes
Authors: Ko Rinkei , Smith Linda Tombras
Published: December 2019

The ability of the gram-positive, food-borne pathogen
Listeria monocytogenes
to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na
+
-glycine betaine symport. In this report, we identify a second glycine betaine transporter from
L. monocytogenes
which is osmotically activated but does not require a high concentration of Na
+
for activity. By using a pool of Tn
917
-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to
opuA
from
Bacillus subtilis
and
proU
from
Escherichia coli
, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both
opuA
and
proU
. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in
L. monocytogenes
appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na
+
is an indication that only the ATP-dependent transporter and the Na
+
-glycine betaine symporter occur in
L. monocytogenes
.
Oxygen-Sensing Reporter Strain of
Pseudomonas fluorescens
for Monitoring the Distribution of Low-Oxygen Habitats in Soil
Authors: Højberg Ole , Schnider Ursula , Winteler Harald V. , Sørensen Jan , Haas Dieter
Published: December 2019

The root-colonizing bacterium
Pseudomonas fluorescens
CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of
Pseudomonas aeruginosa
, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural
lacZ
gene of
Escherichia coli
. By inserting the reporter fusion into the chromosomal
att
Tn
7
site of
P. fluorescens
CHA0 by using a mini-Tn
7
transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 × 10
2
Pa to 2 × 10
2
Pa of O
2
by a nearly 100-fold increase in β-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 × 10
2
Pa. This dose response closely resembles that found for
E. coli
promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.
Bactericidal Activity of Photocatalytic TiO
2
Reaction: toward an Understanding of Its Killing Mechanism
Authors: Maness Pin-Ching , Smolinski Sharon , Blake Daniel M. , Huang Zheng , Wolfrum Edward J. , Jacoby William A.
Published: December 2019

When titanium dioxide (TiO
2
) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of
Escherichia coli
K-12 cells that are irradiated in the presence of the TiO
2
photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol · mg (dry weight) of cells
−1
after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO
2
. We concluded that TiO
2
photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the
E. coli
cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.
Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on
Staphylococcus aureus
Protein A
Authors: Gunneriusson Elin , Samuelson Patrik , Ringdahl Jenny , Grönlund Hans , Nygren Per-Åke , Ståhl Stefan
Published: December 2019

An expression system designed for cell surface display of hybrid proteins on
Staphylococcus carnosus
has been evaluated for the display of
Staphylococcus aureus
protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of
S. carnosus
by using translocation signals from a
Staphylococcus hyicus
lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of
S. carnosus
. Furthermore, these chimeric receptors were found to be functional, since recombinant
S. carnosus
cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.