bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from “
Protaminobacter rubrum
”
Authors: Lee Hyeon Cheol , Kim Jin Ha , Kim Sang Yong , Lee Jung Kul 
Published: June 2008

ABSTRACT

“
Protaminobacter rubrum
” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a
325
RLDRD
329
motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg
325
and Arg
328
in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R
325
Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter
−1
and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter
−1
) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g
−1
with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.
Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH
Authors: Hoshino Tatsuhiko , Yilmaz L. Safak , Noguera Daniel R. , Daims Holger , Wagner Michael 
Published: June 2008

ABSTRACT

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for
Escherichia coli
hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per
E. coli
cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).
Emulsifying and Metal Ion Binding Activity of a Glycoprotein Exopolymer Produced byPseudoalteromonassp. Strain TG12
Authors: Gutierrez Tony , Shimmield Tracy , Haidon Cheryl , Black Kenny , Green David H. 
Published: June 2008

ABSTRACTIn this study, we describe the isolation and characterization of a new exopolymer that exhibits high emulsifying activities against a range of oil substrates and demonstrates a differential capacity to desorb various mono-, di-, and trivalent metal species from marine sediment under nonionic and seawater ionic-strength conditions. This polymer, PE12, was produced by a new isolate,Pseudoalteromonassp. strain TG12 (accession number EF685033), during growth in a modified Zobell's 2216 medium amended with 1% glucose. Chemical and chromatographic analysis showed it to be a high-molecular-mass (>2,000 kDa) glycoprotein composed of carbohydrate (32.3%) and protein (8.2%). PE12 was notable in that it contained xylose as the major sugar component at unusually high levels (27.7%) not previously reported for aPseudoalteromonasexopolymer. The polymer was shown to desorb various metal species from marine sediment—a function putatively conferred by its high content of uronic acids (28.7%). Seawater ionic strength (simulated using 0.6 M NaCl), however, caused a significant reduction in PE12's ability to desorb the sediment-adsorbed metals. These results demonstrate the importance of electrolytes, a physical parameter intrinsic of seawater, in influencing the interaction of microbial exopolymers with metal ions. In summary, PE12 may represent a new class ofPseudoalteromonasexopolymer with a potential for use in biotechnological applications as an emulsifying or metal-chelating agent. In addition to the biotechnological potential of these findings, the ecological aspects of this and related bacterial exopolymers in marine environments are also discussed.
Suppressive Effect on Activation of Macrophages by
Lactobacillus casei
Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides
Authors: Yasuda Emi , Serata Masaki , Sako Tomoyuki 
Published: June 2008

ABSTRACT

Although many
Lactobacillus
strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of
Lactobacillus casei
strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of
L. casei
Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to
L. casei
Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of
L. casei
Shirota, which have been named
cps1A
,
cps1B
,
cps1C
,
cps1D
,
cps1E
,
cps1F
,
cps1G
, and
cps1J
, determine the synthesis of the high-molecular-mass polysaccharide moiety of the
L. casei
Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by
L. casei
Shirota.
Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State
Authors: Dimitriu Pedro A. , Pinkart Holly C. , Peyton Brent M. , Mormile Melanie R. 
Published: June 2008

ABSTRACT

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by
Gammaproteobacteria
, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the
Cytophaga-Flexibacter-Bacteroides
(CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the
Chloroflexi
and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.
Transfer of Class 1 Integron-Mediated Antibiotic Resistance Genes from Shiga Toxin-Producing
Escherichia coli
to a Susceptible
E. coli
K-12 Strain in Storm Water and Bovine Feces
Authors: Nagachinta Supakana , Chen Jinru 
Published: June 2008

ABSTRACT

Transfer of class 1 integron-mediated antibiotic resistance genes has been demonstrated under laboratory conditions. However, there is no information concerning the transfer of these genes in an agricultural environment. The present study sought to determine if integron-mediated streptomycin and sulfisoxazole resistance genes could be transferred from Shiga toxin-producing
Escherichia coli
(STEC) strains 6-20 (O157:H7) and 7-63 (O111:H8) to the susceptible strain
E. coli
K-12 MG1655 in bovine feces (pH 5.5, 6.0, or 6.5) and storm water (pH 5, 6, 7, or 8) at 4, 15, and 28°C, which are average seasonal temperatures for winter, spring-fall, and summer, respectively, in the Griffin, GA, area. The results indicated that at 28°C, the integron-mediated antibiotic resistance genes were transferred from both of the STEC donors in bovine feces. Higher conjugation efficiencies were, however, observed in the conjugation experiments involving STEC strain 6-20. In storm water, the resistance genes were transferred only from STEC strain 6-20. Greater numbers of transconjugants were recovered in the conjugation experiments performed with pH 6.5 bovine feces and with pH 7 storm water. Antibiotic susceptibility tests confirmed the transfer of integron-mediated streptomycin resistance and sulfisoxazole resistance, as well as the transfer of non-integron-mediated oxytetracycline resistance and tetracycline resistance in the transconjugant cells. These results suggest that the antibiotic resistance genes in STEC could serve as a source of antibiotic resistance genes disseminated via conjugation to susceptible cells of other
E. coli
strains in an agricultural environment.
Production of an Insecticidal Crystal Protein from
Bacillus thuringiensis
by the Methylotroph
Methylobacterium extorquens
Authors: Choi Young J. , Gringorten J. Lawrence , Bélanger Louise , Morel Lyne , Bourque Denis , Masson Luke , Groleau Denis , Míguez Carlos B. 
Published: June 2008

ABSTRACT

The Cry1Aa protein from
Bacillus thuringiensis
is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the
cry1Aa
gene in a plant-colonizing methylotroph,
Methylobacterium extorquens
, under the control of the strong
M. extorquens
AM1 methanol dehydrogenase promoter, P

mxaF

. Transmission electron microscopy revealed characteristic bipyramidal intracellular δ-endotoxin crystals similar to the crystalline inclusions formed by
B. thuringiensis
. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm,
Bombyx mori
, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total
M. extorquens
cell protein. The potential benefits of modifying
M. extorquens
to deliver insecticidal Cry proteins for crop and forest protection are discussed.
Function of Heterologous
Mycobacterium tuberculosis
InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C
20
Fatty Acids to C
60
-to-C
90
Mycolic Acids, during De Novo Lipoic Acid Synthesis in
Saccharomyces cerevisiae
Authors: Gurvitz Aner , Hiltunen J. Kalervo , Kastaniotis Alexander J. 
Published: June 2008

ABSTRACT

We describe the physiological function of heterologously expressed
Mycobacterium tuberculosis
InhA during de novo lipoic acid synthesis in yeast (
Saccharomyces cerevisiae
) mitochondria. InhA, representing 2-
trans
-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C
20
fatty acids to form C
60
-to-C
90
mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in
S. cerevisiae etr1
Δ cells lacking mitochondrial 2-
trans
-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast
etr1
Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C
4
to C
8
) than was previously thought (>C
12
). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in
M. tuberculosis
and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.
Characterization of Verotoxin-Encoding Phages from
Escherichia coli
O103:H2 Strains of Bovine and Human Origins
Authors: Karama Musafiri , Gyles Carlton L. 
Published: June 2008

ABSTRACT

The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing
Escherichia coli
(VTEC) O103:H2 strains of human and bovine origins. All the strains carried the
vt1
gene, and two carried the
vt2
gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and
Q
and
P
genes, characteristic of lambdoid phages. A total of 32
vt
-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were
vt1
positive, and two were
vt2
positive. Ten of the 30
vt1
-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two
vt2
-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The
Q
and
P
genes were amplified in all 32 phages, and the
Q
-

stxA
1

gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.
Characterization of an
Escherichia coli
O157:H7 O-Antigen Deletion Mutant and Effect of the Deletion on Bacterial Persistence in the Mouse Intestine and Colonization at the Bovine Terminal Rectal Mucosa
Authors: Sheng Haiqing , Lim Ji Youn , Watkins Maryann K. , Minnich Scott A. , Hovde Carolyn J. 
Published: June 2008

ABSTRACT

Escherichia coli
O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an
E. coli
O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (
per
) in the
rfb
cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Δ
per
mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Δ
per
mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Δ
per
mutant shed lower numbers of bacteria (
P
< 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Δ
per
mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (
P
< 0.05). Our previous work showed that flagella do not influence
E. coli
O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.
Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by
Carnobacterium maltaromaticum
UAL307
Authors: Martin-Visscher Leah A. , van Belkum Marco J. , Garneau-Tsodikova Sylvie , Whittal Randy M. , Zheng Jing , McMullen Lynn M. , Vederas John C. 
Published: June 2008

ABSTRACT

Carnobacterium maltaromaticum
UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous
Listeria
species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by
Carnobacterium
spp.
Isolation of Microbes from Lake Vostok Accretion Ice
Authors: D'Elia Tom , Veerapaneni Ram , Rogers Scott O. 
Published: June 2008

ABSTRACT
Bacteria from seven Lake Vostok accretion and two deep glacial Vostok ice core sections were characterized. The cell concentrations were low, but many of the cells were viable. From the hundreds of cultures, 18 unique bacterial rRNA gene phylotypes were determined. Lake Vostok may contain a complex microbial ecosystem.
Magnetic Resonance Imaging of Structure, Diffusivity, and Copper Immobilization in a Phototrophic Biofilm
Authors: Phoenix V. R. , Holmes W. M. 
Published: June 2008

ABSTRACT

Magnetic resonance imaging (MRI) was used to spatially resolve the structure, water diffusion, and copper transport of a phototrophic biofilm and its fate. MRI was able to resolve considerable structural heterogeneity, ranging from classical laminations ∼500 μm thick to structures with no apparent ordering. Pulsed-field gradient (PFG) analysis spatially resolved water diffusion coefficients which exhibited relatively little or no attenuation (diffusion coefficients ranged from 1.7 × 10
−9
m
2
s
−1
to 2.2 × 10
−9
m
2
s
−1
). The biofilm was then reacted with a 10-mg liter
−1
Cu
2+
solution, and transverse-parameter maps were used to spatially and temporally map copper immobilization within the biofilm. Significantly, a calibration protocol similar to that used in biomedical research successfully quantified copper concentrations throughout the biofilm. Variations in Cu concentrations were controlled by the biofilm structure. Copper immobilization was most rapid (∼5 mg Cu liter
−1
h
−1
) over the first 20 to 30 h and then much slower for the remaining 60 h of the experiment. The transport of metal within the biofilm is controlled by both diffusion and immobilization. This was explored using a Bartlett and Gardner model which examined both diffusion and adsorption through a hypothetical film exhibiting properties similar to those of the phototrophic biofilm. Higher adsorption constants (
K
) resulted in longer lag times until the onset of immobilization at depth but higher actual adsorption rates. MRI and reaction transport models are versatile tools which can significantly improve our understanding of heavy metal immobilization in naturally occurring biofilms.