bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Field-Based Stable Isotope Probing To Identify Adapted Populations and Track Carbon Flow through a Phenol-Degrading Soil Microbial Community
Authors: DeRito Christopher M. , Pumphrey Graham M. , Madsen Eugene L. 
Published: December 2005

ABSTRACT

The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured
13
CO
2
respired from [
13
C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that
13
C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [
13
C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [
13
C]phenol, and (iii) 12 daily doses of [
13
C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted
13
CO
2
evolution by a factor of 10. Furthermore, imaging of
13
C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated
13
C into their biomass. PCR amplification and 16S rRNA gene sequencing of
13
C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera
Kocuria
and
Staphylococcus
, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus
Pseudomonas
. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.
Modeling Surface Growth of
Escherichia coli
on Agar Plates
Authors: Fujikawa Hiroshi , Morozumi Satoshi 
Published: December 2005

ABSTRACT

Surface growth of
Escherichia coli
cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H. Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.
Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification
Authors: Maruyama Fumito , Kenzaka Takehiko , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao 
Published: December 2005

ABSTRACTRolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count singleEscherichia colicells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes insideE. colicells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA.E. colicells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.
Triphenylmethane Reductase from
Citrobacter
sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in
Escherichia coli
Authors: Jang Moon-Sun , Lee Young-Mi , Kim Cheorl-Ho , Lee Jai-Heon , Kang Dong-Woo , Kim Seok-Jo , Lee Young-Choon 
Published: December 2005

ABSTRACT

We purified to homogeneity an enzyme from
Citrobacter
sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from
Listeria monocytogenes
strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in
Escherichia coli
, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.
l-Selective Amidase with Extremely Broad Substrate Specificity fromOchrobactrum anthropiNCIMB 40321
Authors: Sonke Theo , Ernste Sandra , Tandler Renate F. , Kaptein Bernard , Peeters Wilco P. H. , van Assema Friso B. J. , Wubbolts Marcel G. , Schoemaker Hans E. 
Published: December 2005

ABSTRACTAn industrially attractivel-specific amidase was purified to homogeneity fromOchrobactrum anthropiNCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60°C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+(to 80%), Mn2+(to 400%), and Mg2+(to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of α-hydrogen- and (bulky) α,α-disubstituted α-amino acid amides, α-hydroxy acid amides, and α-N-hydroxyamino acid amides. In all cases, only thel-enantiomer was hydrolyzed, resulting inEvalues of more than 150. Simple aliphatic amides, β-amino and β-hydroxy acid amides, and dipeptides were not converted. The gene encoding thisl-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression inEscherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.
Molecular Analysis of the O-Antigen Gene Cluster ofEscherichia coliO86:B7 and Characterization of the Chain Length Determinant Gene (wzz)
Authors: Guo Hongjie , Yi Wen , Shao Jun , Lu Yuquan , Zhang Wenpeng , Song Jing , Wang Peng George 
Published: December 2005

ABSTRACTEscherichia coliO86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster betweengalFandhisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions betweengalFandgndare identical, includingwzygenes. However, deletion of the twowzygenes revealed thatwzyin O86:B7 is responsible for the polymerization of the O antigen, while the deletion ofwzyin O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functionalwzygene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that thewzzgenes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.
Exogenous Isoleucine and Fatty Acid Shortening Ensure the High Content of Anteiso-C
15:0
Fatty Acid Required for Low-Temperature Growth of
Listeria monocytogenes
Authors: Zhu Kun , Ding Xiang , Julotok Mudcharee , Wilkinson Brian J. 
Published: December 2005

ABSTRACT

Previous studies have demonstrated that the branched-chain fatty acid anteiso-C
15:0
plays a critical role in the growth of
Listeria monocytogenes
at low temperatures by ensuring sufficient membrane fluidity. Studies utilizing a chemically defined minimal medium revealed that the anteiso fatty acid precursor isoleucine largely determined the fatty acid profile and fatty acid response of the organism to lowered growth temperature. When isoleucine was sufficient, the fatty acid profile was very uniform, with anteiso fatty acids comprising up to 95% of total fatty acid, and the major fatty acid adjustment to low temperature was fatty acid chain shortening, which resulted in an increase of anteiso-C
15:0
solely at the expense of anteiso-C
17:0
. When isoleucine was not supplied, the fatty acid profile became more complex and was readily modified by leucine, which resulted in a significant increase of corresponding iso fatty acids and an inability to grow at 10°C. Under this condition, the increase of anteiso-C
15:0
at low temperature resulted from the combined effect of increasing the anteiso:iso ratio and chain shortening. A branched-chain α-keto acid dehydrogenase-defective strain largely lost the ability to increase the anteiso:iso ratio. Cerulenin, an inhibitor of β-ketoacyl-acyl carrier protein synthase (FabF), induced a similar fatty acid chain shortening as low temperature did. We propose that the anteiso precursor preferences of enzymes in the branched-chain fatty acid biosynthesis pathway ensure a high production of anteiso fatty acids, and cold-regulated chain shortening results in a further increase of anteiso-C
15:0
at the expense of anteiso-C
17:0
.
Differential Binding of
Escherichia coli
O157:H7 to Alfalfa, Human Epithelial Cells, and Plastic Is Mediated by a Variety of Surface Structures
Authors: Torres Alfredo G. , Jeter Cecelia , Langley William , Matthysse Ann G. 
Published: December 2005

ABSTRACT

Escherichia coli
O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several
E. coli
O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (
cah
,
aidA1
, and
ompA
) and mediators of hyperadherence (
tdcA
,
yidE
,
waaI
, and
cadA
) and those associated with fimbria formation (
csgA
,
csgD
, and
lpfD2
). The introduction of some of these genes (
cah
,
aidA1
, and
csg
loci) into an
E. coli
K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of
E. coli
O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the
ompA
gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a
tdcA ompA
double mutant. In contrast, the
E. coli
O157:H7
ompA
and
tdcA ompA
mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in
E. coli
O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was
ompA
.
Compatible Solutes of the HyperthermophilePalaeococcus ferrophilus: Osmoadaptation and Thermoadaptation in the OrderThermococcales
Authors: Neves Clélia , da Costa Milton S. , Santos Helena 
Published: December 2005

ABSTRACTThe effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeonPalaeococcus ferrophilus. The genusPalaeococcusrepresents a deep-branching lineage of the orderThermococcales, which diverged beforeThermococcusandPyrococcus. Palaeococcus ferrophilusaccumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the generaPyrococcusandThermococcus,Palaeococcus ferrophilusdid not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate inPalaeococcus ferrophilusandThermococcus litoraliswere identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). ThempgSandmpgPgenes ofPalaeococcus ferrophiluswere expressed inEscherichia coliand the proteins were characterized. MPGS had maximal activity at 90°C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90°C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83°C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of theThermococcalesis presented.
Relatedness of
Listeria monocytogenes
Isolates Recovered from Selected Ready-To-Eat Foods and Listeriosis Patients in the United States
Authors: Gilbreth Stefanie Evans , Call Jeff E. , Wallace F. Morgan , Scott Virginia N. , Chen Yuhuan , Luchansky John B. 
Published: December 2005

ABSTRACT

Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of
Listeria monocytogenes
, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, ≥66%), 139 AscI pulsotypes (levels of relatedness, ≥25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the
L. monocytogenes
subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.
Survival of Spacecraft-Associated Microorganisms under Simulated Martian UV Irradiation
Authors: Newcombe David A. , Schuerger Andrew C. , Benardini James N. , Dickinson Danielle , Tanner Roger , Venkateswaran Kasthuri 
Published: December 2005

ABSTRACT

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three
Bacillus
spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m
−2
of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of
Bacillus
species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain,
Bacillus subtilis
168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the
Bacillus
species tested, spores of a
Bacillus pumilus
strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific;
B. pumilus
SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like
B. pumilus
SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.
Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of
Mycobacterium tuberculosis
?
Authors: Gibson Andrea , Brown Timothy , Baker Lucy , Drobniewski Francis 
Published: December 2005

ABSTRACT

The phylogeny and evolution of the bacterium
Mycobacterium tuberculosis
is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469
M. tuberculosis
and 49
Mycobacterium bovis
isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the
M. tuberculosis
complex. This population was characterized by previously identified silent single-nucleotide polymorphisms (sSNPs) or by a macroarray based on these sSNPs that was developed in this study. MIRU-VNTR phylogenetic codes capable of differentiating between phylogenetic lineages were identified. Overall, there was 90.9% concordance between the lineages of isolates as defined by the MIRU-VNTR and sSNP analyses. The MIRU-VNTR phylogenetic code was unique to
M. bovis
and was not observed in any
M. tuberculosis
isolates. The codes were able to differentiate between different
M. tuberculosis
strain families such as Beijing, Delhi, and East African-Indian. Discrepant isolates with similar but not identical MIRU-VNTR codes often displayed a stepwise trend suggestive of bidirectional evolution. A lineage-specific panel of MIRU-VNTR can be used to subdivide each lineage for epidemiological purposes. MIRU-VNTR is a valuable tool for phylogenetic studies and could define an evolutionarily uncharacterized population of
M. tuberculosis
complex organisms.