bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of ArfI, an α-
l
-Arabinofuranosidase from
Cytophaga xylanolytica
Authors: Renner Michael J. , Breznak John A. 
Published: December 2019

ABSTRACT

An α-
l
-arabinofuranosidase (α-
l
-arabinofuranoside arabinofuranohydrolase [EC
3.2.1.55
]; referred to below as ArfI) from
Cytophaga xylanolytica
XM3 was purified 85-fold by anion-exchange and hydrophobic interaction column chromatography. The native enzyme had a pI of 6.1 and an apparent molecular mass of 160 to 210 kDa, and it appeared to be a trimer or tetramer consisting of 56-kDa subunits. With
p
-nitrophenyl-α-
l
-arabinofuranoside as the substrate, the enzyme exhibited a

K
m

of 0.504 mM and a
V
max
of 319 μmol · min
−1
· mg of protein
−1
, and it had optimum activity at pH 5.8 and 45°C. ArfI was relatively stable over a pH range of 4 to 10 and at temperatures up to 45°C, and it retained nearly full activity when stored at 4°C for periods as long as 24 months. The enzyme also released arabinose from 4-methylumbelliferyl-α-
l
-arabinofuranoside, as well as from rye, wheat, corn cob, and oat spelt arabinoxylans and sugar beet arabinan, but not from arabinogalactan. ArfI showed no hydrolytic activity toward a range of
p
-nitrophenyl- or 4-methylumbelliferyl-glycosides other than arabinoside, for which it was entirely specific for the α-
l
-furanoside configuration. ArfI interacted synergistically with three partially purified endoxylanase fractions from
C. xylanolytica
in hydrolyzing rye arabinoxylan. However, cell fractionation studies revealed that ArfI was largely, if not entirely, cytoplasmic, so its activity in vivo is probably most relevant to hydrolysis of arabinose-containing oligosaccharides small enough to pass through the cytoplasmic membrane. Antibodies prepared against purified ArfI also cross-reacted with arabinofuranosidases from other freshwater and marine strains of
C. xylanolytica
, as well as with some proteins that did not possess arabinofuranosidase activity. To our knowledge, this is the first α-
l
-arabinofuranosidase to be purified and characterized from any gliding bacterium.
IS
D1
, an Insertion Element from the Sulfate-Reducing Bacterium
Desulfovibrio vulgaris
Hildenborough: Structure, Transposition, and Distribution
Authors: Fu Rongdian , Voordouw Gerrit 
Published: December 2019

ABSTRACT

Insertion element IS
D1
, discovered when its transposition caused the insertional inactivation of an introduced
sacB
gene, is present in two copies in the genome of
Desulfovibrio vulgaris
Hildenborough. Southern blot analysis indicated at least two insertion sites in the
sacB
gene. Cloning and sequencing of a transposed copy of IS
D1
indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. IS
D1
encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A
6
G shifty codon motif. Sequence comparison showed that IS
D1
belongs to the IS
3
family. Isolation and analysis of the chromosomal copies, IS
D1
-A and IS
D1
-B, by PCR and sequencing indicated that these are not flanked by direct repeats. IS
D1
-A is inserted in a region of the chromosome containing the
gapdh-pgk
genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. IS
D1
is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of IS
D1
in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of
D. desulfuricans
Norway.
Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized byCeriporiopsis subvermisporaProduces Extracellular Hydrogen Peroxide
Authors: Urzúa Ulises , Kersten Philip J. , Vicuña Rafael 
Published: December 2019

ABSTRACTThe ligninolytic system of the basidiomyceteCeriporiopsis subvermisporais composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid ofC. subvermisporacultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured;14CO2evolution was monitored after addition of exogenous [14C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO2from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [14C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [14C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide byC. subvermispora.
Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by
Thermococcus
spp
Authors: Lamosa Pedro , Martins Lígia O. , Da Costa Milton S. , Santos Helena 
Published: December 2019

ABSTRACT

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in
Thermococcus litoralis
,
Thermococcus celer
,
Thermococcus stetteri
, and
Thermococcus zilligii
(strain AN1). In addition, the effects of growth stage and composition of the medium were studied in
T. litoralis
. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in
T. litoralis
grown on peptone-containing medium. Besides this newly discovered compound,
T. litoralis
accumulated mannosylglycerate, aspartate, α-glutamate, di-
myo
-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other
Thermococcus
strains examined. Di-
myo
-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in
T. celer
and
T. stetteri
, and the latter organism also accumulated α-glutamate. The only nonmarine species studied,
T. zilligii
, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in
T. litoralis
,
T. celer
, and
T. stetteri
in response to salt stress, while di-
myo
-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in
T. litoralis
; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-
myo
-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of
T. litoralis
to scavenge suitable components from the medium and to use them as compatible solutes.
Analysis of the Gene Cluster Encoding Toluene/
o
-Xylene Monooxygenase from
Pseudomonas stutzeri
OX1
Authors: Bertoni Giovanni , Martino Manuela , Galli Enrica , Barbieri Paola 
Published: December 2019

ABSTRACT

The toluene/
o
-xylene monooxygenase cloned from
Pseudomonas stutzeri
OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from
Pseudomonas
strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/
o
-xylene monooxygenase in
Escherichia coli
. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/
o
-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.
Effect of Butyrolactone I on the Producing Fungus,
Aspergillus terreus
Authors: Schimmel Timothy G. , Coffman Allen D. , Parsons Sarah J. 
Published: December 2019

ABSTRACT

Butyrolactone I [α-oxo-β-(
p
-hydroxyphenyl)-γ-(
p
-hydroxy-
m
-3,3-dimethylallyl-benzyl)-γ-methoxycarbonyl-γ-butyrolactone] is produced as a secondary metabolite by
Aspergillus terreus
. Because small butyrolactone-containing molecules act as self-regulating factors in some bacteria, the effects of butyrolactone I on the producing organism were studied; specifically, changes in morphology, sporulation, and secondary metabolism were studied. Threefold or greater increases in hyphal branching (with concomitant decreases in the average hyphal growth unit), submerged sporulation, and secondary metabolism were observed when butyrolactone I was added to cultures of
A. terreus
. Among the secondary metabolites whose production was increased by this treatment was the therapeutically important compound lovastatin. These findings indicate that butyrolactone I induces morphological and sporulation changes in
A. terreus
and enhances secondary metabolite production in a manner similar to that previously reported for filamentous bacteria.
Bias in Template-to-Product Ratios in Multitemplate PCR
Authors: Polz Martin F. , Cavanaugh Colleen M. 
Published: December 2019

ABSTRACT
Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450
BM-3
Monooxygenase
Authors: Schneider Silke , Wubbolts Marcel G. , Sanglard Dominique , Witholt Bernard 
Published: December 2019

ABSTRACT

The application of whole cells containing cytochrome P-450
BM-3
monooxygenase [EC
1.14.14.1
] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose,
Escherichia coli
recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450
BM-3
were used. To overcome inefficient uptake of pentadecanoic acid by intact
E. coli
cells, we made use of a cloned fatty acid uptake system from
Pseudomonas oleovorans
which, in contrast to the common FadL fatty acid uptake system of
E. coli
, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from
P. oleovorans
is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in
E. coli
W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of
E. coli
K27, which is a
fadD
mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450
BM-3
monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from
P. oleovorans
, cytochrome P-450
BM-3
recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.
Manganese Reduction by Microbes from Oxic Regions of the Lake Vanda (Antarctica) Water Column
Authors: Bratina Bonnie Jo , Stevenson Bradley S. , Green William J. , Schmidt Thomas M. 
Published: December 2019

ABSTRACT

Depth profiles of metals in Lake Vanda, a permanently ice-covered, stratified Antarctic lake, suggest the importance of particulate manganese oxides in the scavenging, transport, and release of metals. Since manganese oxides can be solubilized by manganese-reducing bacteria, microbially mediated manganese reduction was investigated in Lake Vanda. Microbes concentrated from oxic regions of the water column, encompassing a peak of soluble manganese [Mn(II)], reduced synthetic manganese oxides (MnO
2
) when incubated aerobically. Pure cultures of manganese-reducing bacteria were readily isolated from waters collected near the oxic Mn(II) peak. Based on phylogenetic analysis of the 16S rRNA gene sequence, most of the isolated manganese reducers belong to the genus
Carnobacterium
. Cultures of a phylogenetically representative strain of
Carnobacterium
reduced synthetic MnO
2
in the presence of sodium azide, as was seen in field assays. Unlike anaerobes that utilize manganese oxides as terminal electron acceptors in respiration, isolates of the genus
Carnobacterium
reduced Mn(IV) via a diffusible compound under oxic conditions. The release of adsorbed trace metals accompanying the solubilization of manganese oxides may provide populations of
Carnobacterium
with a source of nutrients in this extremely oligotrophic environment.
Microbial Diversity in a Hydrocarbon- and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation
Authors: Dojka Michael A. , Hugenholtz Philip , Haack Sheridan K. , Pace Norman R. 
Published: December 2019

ABSTRACT

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or
Bacteria
- or
Archaea
-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus
Syntrophus
;
Syntrophus
spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to
Methanosaeta
spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.
Stereo- and Regioselective Hydroxylation of α-Ionone by
Streptomyces
Strains
Authors: Lutz-Wahl Sabine , Fischer Peter , Schmidt-Dannert Claudia , Wohlleben Wolfgang , Hauer Bernhard , Schmid Rolf D. 
Published: December 2019

ABSTRACT

A total of 215
Streptomyces
strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15
Streptomyces
strains,
S. fradiae
Tü27,
S. arenae
Tü 495,
S. griseus
ATCC 13273,
S. violaceoniger
Tü 38, and
S. antibioticus
Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6
R
)-(−)/(6
S
)-(+)] resulted in the exclusive formation of only the two enantiomers (3
R
,6
R
)- and (3
S
,6
S
)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the
Streptomyces
strains tested proceeds with both high regio- and stereoselectivity.
Molecular Characterization of Plasmid-Mediated Oxytetracycline Resistance in
Aeromonas salmonicida
Authors: Adams C. A. , Austin B. , Meaden P. G. , McIntosh D. 
Published: December 2019

ABSTRACT

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of
Aeromonas salmonicida
transferred the OT resistance phenotype to
Escherichia coli
. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of
Aeromonas
spp. The molecular basis for antibiotic resistance in OT-resistant isolates of
A. salmonicida
was determined. The OT resistance determinant from one plasmid (pASOT) of
A. salmonicida
was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb
Eco
RI fragment on R-plasmids from the 19 OT-resistant isolates of
A. salmonicida
. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19
A. salmonicida
isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.
Monoclonal Antibodies Raised against Native Major Capsid Proteins of Lactococcal c2-Like Bacteriophages
Authors: Chibani Azaïez Sandra R. , Fliss Ismaïl , Simard Ronald E. , Moineau Sylvain 
Published: December 2019

ABSTRACT

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a
Lactococcus lactis
cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.
Physiological Diversity of the Rhizosphere Diazotroph Assemblages of Selected Salt Marsh Grasses
Authors: Bagwell Christopher E. , Piceno Yvette M. , Ashburne-Lucas Amy , Lovell Charles R. 
Published: December 2019

ABSTRACT

Rhizosphere diazotroph assemblages of salt marsh grasses are thought to be influenced by host plant species and by a number of porewater geochemical parameters. Several geochemical variables can adversely affect plant productivity and spatial distributions, resulting in strong zonation of plant species and growth forms. This geochemically induced stress may also influence the species compositions and distributions of rhizosphere diazotroph assemblages, but little is currently known about these organisms. The diversity and key physiological features of culturable, O
2
-tolerant rhizosphere diazotrophs associated with the tall and short growth forms of
Spartina alterniflora
and with
Juncus roemerianus
were examined. A total of 339 gram-negative strains were isolated by a root stab culture approach and morphologically and physiologically characterized by using API and BIOLOG tests. Eighty-six distinct groups composed of physiologically similar strains were identified. Of these groups, 72% were shown to be capable of N
2
fixation through molecular analyses, and a representative strain was chosen from each diazotroph group for further characterization. Cluster and principal-components analysis of BIOLOG data allowed the designation of physiologically distinct strain groupings. Most of these groups were dominated by strains that were not identifiable to species on the basis of API or BIOLOG testing. Representatives of several families including the
Enterobacteriaceae
,
Vibrionaceae
,
Azotobacteraceae
,
Spirillaceae
,
Pseudomonadaceae
, and
Rhizobiaceae
were recovered, as well as strains with no clear taxonomic affiliations. This study identifies numerous potentially important physiological groups of the salt marsh diazotroph assemblage.