bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of EnterotoxigenicClostridium perfringensType A Isolates in American Retail Foods
Authors: Wen Qiyi , McClane Bruce A.
Published: May 2004

Currently there is only limited understanding of the reservoirs forClostridium perfringenstype A food poisoning. A recent survey (Y.-T. Lin and R. Labbe, Appl. Environ. Microbiol.69:1642-1646, 2003) of non-outbreak American retail foods did not identify the presence of a singleC. perfringensisolate carrying the enterotoxin gene (cpe) necessary for causing food poisoning. The present study revisited this issue, using revised methodology and food sampling strategies. In our survey,cpe-positiveC. perfringensisolates were detected in ∼1.4% of ∼900 surveyed non-outbreak American retail foods. Interestingly, those enterotoxigenic isolates in non-outbreak foods appear indistinguishable fromC. perfringensisolates known to cause food poisoning outbreaks: i.e., the enterotoxigenic retail food isolates all carry a chromosomalcpegene, are classified as type A, and exhibit exceptional heat resistance. Collectively, these findings indicate that some American foods are contaminated, at the time of retail purchase, withC. perfringensisolates having full potential to cause food poisoning. Furthermore, demonstrating that type A isolates carrying a chromosomalcpegene are the enterotoxigenic isolates most commonly present in foods helps to explain why these isolates (rather than type A isolates carrying a plasmidcpegene orcpe-positive type C or D isolates) are strongly associated with food poisoning outbreaks. Finally, since type A chromosomalcpeisolates present in the surveyed raw foods exhibited strong heat resistance, it appears that exceptional heat resistance is not a survivor trait selected for by cooking but is instead an intrinsic trait possessed by many type A chromosomalcpeisolates.
Responses of Active Bacterial and Fungal Communities in Soils under Winter Wheat to Different Fertilizer and Pesticide Regimens
Authors: Girvan Martina S. , Bullimore Juliet , Ball Andrew S. , Pretty Jules N. , Osborn A. Mark
Published: May 2004

The composition of the active microbial (bacterial and fungal) soil community in an arable wheat field subjected to different management practices was examined at five times during a 1-year period. Field sections were fertilized either at good agricultural practice (GAP) levels or at reduced levels (0.5× GAP) and were inoculated with vesicular arbuscular mycorrhizae (VAM) at the same time. Field subsections were treated either with or without pesticides. Changes in the active microbial communities were investigated by denaturing gradient gel electrophoresis analysis of reverse transcription-PCR-amplified 16S and 18S rRNA. Microbial community structure was primarily determined by season, and the seasonal trends were similar for the fungal and bacterial components. Between-sample microbial heterogeneity decreased under a mature crop in the summer but increased following harvesting and plowing. Although similar overall trends were seen for the two microbial components, sample variability was greater for the fungal community than for the bacterial community. The greatest management effects were due to GAP fertilization, which caused increases in the bacterial numbers in the total and culturable communities. Microbial biomass similarly increased. GAP fertilization also caused large shifts in both the active bacterial community structure and the active fungal community structure and additionally resulted in a decrease in the heterogeneity of the active bacterial community. Pesticide addition did not significantly affect bacterial numbers or heterogeneity, but it led to major shifts in the active soil bacterial community structure. PCR primers specific for Glomales 25S rRNA genes were used to monitor the VAM population following inoculation. Glomales were detected initially only in VAM-inoculated field sections but were subsequently detected in noninoculated field sections as the season progressed. After plowing, the level of Glomales was reduced in noninoculated field sections but remained high in VAM-inoculated field sections. Inoculation of VAM correlated with elevated soil phosphate and carbon levels.
Behavior of an
Aeromonas hydrophila aroA
Live Vaccine in Water Microcosms
Authors: Vivas José , Carracedo Begoña , Riaño Jorge , Razquin Blanca E. , López-Fierro Pilar , Acosta Félix , Naharro Germán , Villena Alberto J.
Published: May 2004

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an
aroA
mutant of
Aeromonas hydrophila
and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The
aroA
mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the
aroA
strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain
A. hydrophila
AG2; after long periods of incubation,
aroA
cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two
A. hydrophila
strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the
aroA
strain in natural environments and suggest that the inactivation of the
aroA
gene may be beneficial for the safety of this live vaccine for aquacultures.
Modification of Spatial Distribution of 2,4-Dichlorophenoxyacetic Acid Degrader Microhabitats during Growth in Soil Columns
Authors: Pallud C. , Dechesne A. , Gaudet J. P. , Debouzie D. , Grundmann G. L.
Published: May 2004

Bacterial processes in soil, including biodegradation, require contact between bacteria and substrates. Knowledge of the three-dimensional spatial distribution of bacteria at the microscale is necessary to understand and predict such processes. Using a soil microsampling strategy combined with a mathematical spatial analysis, we studied the spatial distribution of 2,4-dichlorophenoxyacetic acid (2,4-D) degrader microhabitats as a function of 2,4-D degrader abundance. Soil columns that allowed natural flow were percolated with 2,4-D to increase the 2,4-D degrader abundance. Hundreds of soil microsamples (minimum diameter, 125 μm) were collected and transferred to culture medium to check for the presence of 2,4-D degraders. Spatial distributions of bacterial microhabitats were characterized by determining the average size of colonized soil patches and the average number of patches per gram of soil. The spatial distribution of 2,4-D degrader microhabitats was not affected by water flow, but there was an overall increase in colonized patch sizes after 2,4-D amendment; colonized microsamples were dispersed in the soil at low 2,4-D degrader densities and clustered in patches that were more than 0.5 mm in diameter at higher densities. During growth, spreading of 2,4-D degraders within the soil and an increase in 2,4-D degradation were observed. We hypothesized that spreading of the bacteria increased the probability of encounters with 2,4-D and resulted in better interception of the degradable substrate. This work showed that characterization of bacterial microscale spatial distribution is relevant to microbial ecology studies. It improved quantitative bacterial microhabitat description and suggested that sporadic movement of cells occurs. Furthermore, it offered perspectives for linking microbial function to the soil physicochemical environment.
Increased ATPase Activity Is Responsible for Acid Sensitivity of Nisin-Resistant
Listeria monocytogenes
ATCC 700302
Authors: McEntire Jennifer Cleveland , Carman George M. , Montville Thomas J.
Published: May 2004

The growth of the foodborne pathogen
Listeria monocytogenes
can be controlled by nisin, an antimicrobial peptide. A spontaneous mutant of
L. monocytogenes
shows both resistance to nisin and increased acid sensitivity compared to the wild type. Changes in the cell membrane correlated with nisin resistance, but the mechanism for acid sensitivity appears unrelated. When hydrochloric or lactic acid is added to cultures, intracellular ATP levels drop significantly in the mutant (
P
< 0.01) compared to the results seen with the wild type. Characterization of the F
0
F
1
ATPase, which hydrolyzes ATP to pump protons from the cell cytoplasm, shows that the enzyme is more active in the mutant than in the wild type. These data support a model in which the increased activity of the mutant ATPase upon acid addition depletes the cells' supply of ATP, resulting in cell death.
Transformation of Verapamil byCunninghamella blakesleeana
Authors: Sun Lu , Huang Hai-Hua , Liu Lei , Zhong Da-Fang
Published: May 2004

A filamentous fungus,Cunninghamella blakesleeanaAS 3.153, was used as a microbial model of mammalian metabolism to transform verapamil, a calcium channel antagonist. The metabolites of verapamil were separated and assayed by the liquid chromatography-ion trap mass spectrometry method. After 96 h of incubation, nearly 93% of the original drug was metabolized to 23 metabolites. Five major metabolites were isolated by semipreparative high-performance liquid chromatography and were identified by proton nuclear magnetic resonance and electrospray mass spectrometry. Other metabolites were characterized according to their chromatographic behavior and mass spectral data. The major metabolic pathways of verapamil transformation by the fungus were N dealkylation, O demethylation, and sulfate conjugation. The phase I metabolites of verapamil (introduction of a functional group) byC. blakesleeanaparalleled those in mammals; therefore,C. blakesleeanacould be a useful tool for generating the mammalian phase I metabolites of verapamil.
Evaluation of a
Clostridium perfringens
Predictive Model, Developed under Isothermal Conditions in Broth, To Predict Growth in Ground Beef during Cooling
Authors: Smith Sarah , Schaffner Donald W.
Published: May 2004

Proper temperature control is essential in minimizing
Clostridium perfringens
germination, growth, and toxin production. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log
10
CFU/g increase of
C. perfringens
and no growth of
Clostridium botulinum
. A mathematical model developed by Juneja et al. (Food Microbiol.
16:
335-349, 1999) may be helpful in determining if the
C. perfringens
performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations. The Juneja 1999 model consistently underpredicted growth of
C. perfringens
during exponential cooling of ground beef. The model also underpredicted growth of
C. perfringens
in ground beef cooled at two different rates. The results presented here show generally good agreement with published data on the growth of
C. perfringens
in similar products. The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.
Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic
Escherichia coli
KO11 during Xylose Fermentation
Authors: Underwood S. A. , Buszko M. L. , Shanmugam K. T. , Ingram L. O.
Published: May 2004

Limited cell growth and the resulting low volumetric productivity of ethanologenic
Escherichia coli
KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml
−1
optical density at 550 nm [OD
550
] unit
−1
), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml
−1
OD
550
unit
−1
), trehalose (9.9 nmol ml
−1
OD
550
unit
−1
), and betaine (19.8 nmol ml
−1
OD
550
unit
−1
). Two independent genetic modifications of
E. coli
KO11 (functional expression of
Bacillus subtilis citZ
encoding NADH-insensitive citrate synthase; deletion of
ackA
encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.
Dissimilatory Arsenate Reduction with Sulfide as Electron Donor: Experiments with Mono Lake Water and Isolation of Strain MLMS-1, a Chemoautotrophic Arsenate Respirer
Authors: Hoeft Shelley E. , Kulp Thomas R. , Stolz John F. , Hollibaugh James T. , Oremland Ronald S.
Published: May 2004

Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H
14
CO
3
−
into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the δ-
Proteobacteria
, located near sulfate reducers like
Desulfobulbus
sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.
Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of
Oenococcus oeni
Authors: Silveira M. Graça , Baumgärtner Maja , Rombouts Frank M. , Abee Tjakko
Published: May 2004

The practical application of commercial malolactic starter cultures of
Oenococcus oeni
surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of
O. oeni
cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol. Levels of inosine-5′-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation. Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EII
Man
, were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to
O. oeni
cells during malolactic fermentation. In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed. Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and
d
-alanine:
d
-alanine ligase, are known to be involved in cell wall biosynthesis. Using a proteomic approach, we provide evidence for an active ethanol adaptation response of
O. oeni
at the cytoplasmic and membrane protein levels.
Subpopulation Characteristics of Egg-Contaminating
Salmonella enterica
serovar Enteritidis as Defined by the Lipopolysaccharide O Chain
Authors: Guard-Bouldin Jean , Gast Richard K. , Humphrey Thomas J. , Henzler David J. , Morales Cesar , Coles Karen
Published: May 2004

Characterization of
Salmonella enterica
serovar Enteritidis was refined by incorporating new data from isolates obtained from avian sources, from the spleens of naturally infected mice, and from the United Kingdom into an existing lipopolysaccharide (LPS) O-chain compositional database. From least to greatest, the probability of avian isolates producing high-molecular-mass LPS O chain ranked as follows: pooled kidney, liver, and spleen; intestine; cecum; ovary and oviduct; albumen; yolk; and whole egg. Mouse isolates were most like avian intestinal samples, whereas United Kingdom isolates were most like those from the avian reproductive tract and egg. Non-reproductive tract organ isolates had significant loss of O chain. Isogenic isolates that varied in ability to make biofilm and to be orally invasive produced different O-chain structures at 25°C but not at 37°C. Hens infected at a 91:9 biofilm-positive/-negative colony phenotype ratio yielded only the negative phenotype from eggs. These results indicate that the environment within the hen applies stringent selection pressure on subpopulations of
S. enterica
serovar Enteritidis at certain points in the infection pathway that ends in egg contamination. The avian cecum, rather than the intestines, is the early interface between the environment and the host that supports emergence of subpopulation diversity. These results suggest that diet and other factors that alter cecal physiology should be investigated as a means to reduce egg contamination.
Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from
Achromobacter xylosoxidans
: Probing the Molecular Basis for Its High Thermostability
Authors: Cai Gang , Zhu Songcheng , Yang Sheng , Zhao Guoping , Jiang Weihong
Published: May 2004

The gene encoding a novel penicillin G acylase (PGA), designated
pgaW
, was cloned from
Achromobacter xylosoxidans
and overexpressed in
Escherichia coli
. The
pgaW
gene contains an open reading frame of 2,586 nucleotides. The deduced protein sequence encoded by
pgaW
has about 50% amino acid identity to several well-characterized PGAs, including those of
Providencia rettgeri
,
Kluyvera cryocrescens
, and
Escherichia coli
. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60°C and its half-life of inactivation at 55°C is four times longer than that of another previously reported thermostable PGA from
Alcaligenes faecalis
(R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.
Two Distinct Pathways for Trehalose Assimilation in the YeastSaccharomyces cerevisiae
Authors: Jules Matthieu , Guillou Vincent , François Jean , Parrou Jean-Luc
Published: May 2004

The yeastSaccharomyces cerevisiaecan synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, theAGT1/MAL11gene is controlled by theMALsystem, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.
Acquisition of an
Agrobacterium
Ri Plasmid and Pathogenicity by Other α-
Proteobacteria
in Cucumber and Tomato Crops Affected by Root Mat
Authors: Weller S. A. , Stead D. E. , Young J. P. W.
Published: May 2004

Root mat of cucumbers and tomatoes has previously been shown to be caused by
Agrobacterium radiobacter
strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring α-
Proteobacteria
have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus
Ochrobactrum
, five as being in the genus
Rhizobium
, and one as being in the genus
Sinorhizobium
. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring α-
Proteobacteria
induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic
Ochrobactrum
(46%) and
Rhizobium
(44%) strains were lower than those observed for rhizogenic
A. radiobacter
strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic
Sinorhizobium
strain (75%).
Detection of Molecular Diversity in
Bacillus atrophaeus
by Amplified Fragment Length Polymorphism Analysis
Authors: Burke S. A. , Wright J. D. , Robinson M. K. , Bronk B. V. , Warren R. L.
Published: May 2004

Phenotypically,
Bacillus atrophaeus
is indistinguishable from the type strain of
Bacillus subtilis
except by virtue of pigment production on certain media. Several pigmented variants of
B. subtilis
have been reclassified as
B. atrophaeus
, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as
Bacillus globigii
,
B. subtilis
var. niger, or
Bacillus niger
using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the
B. atrophaeus
type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to
B. atrophaeus
than to
B. subtilis
and should be reclassified as
B. atrophaeus
. A very closely related cluster of
B. atrophaeus
strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two
B. subtilis
subspecies,
subtilis
and
spizizenii
. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies,
B. atrophaeus
subsp.
globigii
.
Spatial Distribution and Stability of the Eight Microbial Species of the Altered Schaedler Flora in the Mouse Gastrointestinal Tract
Authors: Sarma-Rupavtarm Ramahi B. , Ge Zhongming , Schauer David B. , Fox James G. , Polz Martin F.
Published: May 2004

The overall complexity of the microbial communities in the gastrointestinal (GI) tracts of mammals has hindered observations of dynamics and interactions of individual bacterial populations. However, such information is crucial for understanding the diverse disease-causing and protective roles that gut microbiota play in their hosts. Here, we determine the spatial distribution, interanimal variation, and persistence of bacteria in the most complex defined-flora (gnotobiotic) model system to date, viz., mice colonized with the eight strains of the altered Schaedler flora (ASF). Quantitative PCR protocols based on the 16S rRNA sequence of each ASF strain were developed and optimized to specifically detect as few as 10 copies of each target. Total numbers of the ASF strains were determined in the different regions of the GI tracts of three C.B-17 SCID mice. Individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant
Lactobacillus murinus
ASF361 present at 10
5
to 10
7
cells/g of tissue in the upper GI tract and obligate anaerobic ASF strains being predominant in the cecal and colonic flora at 10
8
to 10
10
cells/g of tissue. The variation between the three mice was small for most ASF strains, except for
Clostridium
sp. strain ASF502 and
Bacteroides
sp. strain ASF519 in the cecum. A comparison of the relative distribution of the ASF strains in feces and the colon indicated large differences, suggesting that fecal bacterial levels may provide a poor approximation of colonic bacterial levels. All ASF strains were detected by PCR in the feces of C57BL/6 restricted flora mice, which had been maintained in an isolator without sterile food, water, or bedding for several generations, providing evidence for the stability of these strains in the face of potential competition by bacteria introduced into the gut.
Double-Layer Plaque Assay for Quantification of Enteroviruses
Authors: Mocé-Llivina Laura , Lucena Francisco , Jofre Juan
Published: May 2004

We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay ≥ suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters ≥ most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2′-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.
New Recombination Methods for
Sinorhizobium meliloti
Genetics
Authors: House Brent L. , Mortimer Michael W. , Kahn Michael L.
Published: May 2004

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium
Sinorhizobium meliloti
in two ways that increase the utility of the system. Adding plasmid
oriT
sequences to a set of vehicles allows the plasmids to be transferred to
S. meliloti
by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of
S. meliloti
, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on
Medicago sativa
(alfalfa) roots.
Transposon Mutagenesis of the Obligate Intracellular Pathogen
Rickettsia prowazekii
Authors: Qin Aiping , Tucker Aimee M. , Hines Andria , Wood David O.
Published: May 2004

Genetic analysis of
Rickettsia prowazekii
has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in
R. prowazekii
and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The
arr-2
gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This
R. prowazekii
-adapted
arr-2
gene (
Rparr-2
) was placed downstream of the strong rickettsial
rpsL
promoter (

rpsL
P

), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing

rpsL
P
-Rparr-2

was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the
R. prowazekii
genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in
R. prowazekii
, thereby identifying nonessential rickettsial genes.
Germination of
Penicillium paneum
Conidia Is Regulated by 1-Octen-3-ol, a Volatile Self-Inhibitor
Authors: Chitarra Gilma S. , Abee Tjakko , Rombouts Frank M. , Posthumus Maarten A. , Dijksterhuis Jan
Published: May 2004

Penicillium paneum
is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions.
P. paneum
produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10
9
conidia ml
−1
. By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25°C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the
P. paneum
life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.
Addition of Aromatic Substrates Restores Trichloroethylene Degradation Activity in
Pseudomonas putida
F1
Authors: Morono Yuki , Unno Hajime , Tanji Yasunori , Hori Katsutoshi
Published: May 2004

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of
Pseudomonas putida
F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.
Prevalence of the
Chloroflexi
-Related SAR202 Bacterioplankton Cluster throughout the Mesopelagic Zone and Deep Ocean
Authors: Morris R. M. , Rappé M. S. , Urbach E. , Connon S. A. , Giovannoni S. J.
Published: May 2004

Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the environmental gene clone cluster known as SAR202 have been recovered from pelagic freshwater, marine sediment, soil, and deep subsurface terrestrial environments. Together, these clones form a major, monophyletic subgroup of the phylum
Chloroflexi
. While members of this diverse group are consistently identified in the marine environment, there are currently no cultured representatives, and very little is known about their distribution or abundance in the world's oceans. In this study, published and newly identified SAR202-related 16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and to design taxon-specific oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda Atlantic time series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean, and along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most abundant below the deep chlorophyll maximum and that they persisted to 3,600 m in the Atlantic Ocean and to 4,000 m in the Pacific Ocean, the deepest samples used in this study. On average, members of the SAR202 group accounted for 10.2% (±5.7%) of all DNA-containing bacterioplankton between 500 and 4,000 m.
Influence of Precipitation and Soil on Transport of Fecal Enterococci in Fractured Limestone Aquifers
Authors: Celico Fulvio , Varcamonti Mario , Guida Marco , Naclerio Gino
Published: May 2004

Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between
Enterococcus faecalis
and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination.
E. faecalis
was chosen because, in the study area involved, it represents a better indicator than
Escherichia coli
. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.
Use of Aqueous Silver To Enhance Inactivation of Coliphage MS-2 by UV Disinfection
Authors: Butkus Michael A. , Labare Michael P. , Starke Jeffrey A. , Moon King , Talbot Mark
Published: May 2004

A synergistic effect between silver and UV radiation has been observed that can appreciably enhance the effectiveness of UV radiation for inactivation of viruses. At a fluence of ca. 40 mJ/cm2, the synergistic effect between silver and UV was observed at silver concentrations as low as 10 μg/liter (P< 0.0615). At the same fluence, an MS-2 inactivation of ca. 3.5 logs (99.97%) was achieved at a silver concentration of 0.1 mg/liter, a significant improvement (P< 0.0001) over the ca. 1.8-log (98.42%) inactivation of MS-2 at ca. 40 mJ/cm2in the absence of silver. Modified Chick-Watson kinetics were used to model the synergistic effect of silver and UV radiation. For an MS-2 inactivation of 4 logs (99.99%), the coefficient of dilution (n) was determined to be 0.31, which suggests that changes in fluence have a greater influence on inactivation than does a proportionate change in silver concentration.
Nitrite Elimination and Hydrolytic Ring Cleavage in 2,4,6-Trinitrophenol (Picric Acid) Degradation
Authors: Hofmann Klaus W. , Knackmuss Hans-Joachim , Heiss Gesche
Published: May 2004

Two hydrogenation reactions in the initial steps of degradation of 2,4,6-trinitrophenol produce the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. The
npdH
gene (contained in the
npd
gene cluster of the 2,4,6-trinitrophenol-degrading strain
Rhodococcus opacus
HL PM-1) was shown here to encode a tautomerase, catalyzing a proton shift between the
aci
-nitro and the nitro forms of the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. An enzyme (which eliminated nitrite from the
aci
-nitro form but not the nitro form of the dihydride complex of 2,4,6-trinitrophenol) was purified from the 2,4,6-trinitrophenol-degrading strain
Nocardioides simplex
FJ2-1A. The product of nitrite release was the hydride Meisenheimer complex of 2,4-dinitrophenol, which was hydrogenated to the dihydride Meisenheimer complex of 2,4-dinitrophenol by the hydride transferase I and the NADPH-dependent F
420
reductase from strain HL PM-1. At pH 7.5, the dihydride complex of 2,4-dinitrophenol is protonated to 2,4-dinitrocyclohexanone. A hydrolase was purified from strain FJ2-1A and shown to cleave 2,4-dinitrocyclohexanone hydrolytically to 4,6-dinitrohexanoate.
Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in
Corynebacterium glutamicum
for Production of Lysine in Whey
Authors: Barrett Eoin , Stanton Catherine , Zelder Oskar , Fitzgerald Gerald , Ross R. Paul
Published: May 2004

The genetic determinants for lactose utilization from
Lactobacillus delbrueckii
subsp.
bulgaricus
ATCC 11842 and galactose utilization from
Lactococcus lactis
subsp.
cremoris
MG 1363 were heterologously expressed in the lysine-overproducing strain
Corynebacterium glutamicum
ATCC 21253. The
C. glutamicum
strains expressing the lactose permease and β-galactosidase genes of
L. delbrueckii
subsp.
bulgaricus
exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly,
C. glutamicum
strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered
C. glutamicum
strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control,
C. glutamicum
21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.
Seasonal Changes in an Alpine Soil Bacterial Community in the Colorado Rocky Mountains
Authors: Lipson David A. , Schmidt Steven K.
Published: May 2004

The period when the snowpack melts in late spring is a dynamic time for alpine ecosystems. The large winter microbial community begins to turn over rapidly, releasing nutrients to plants. Past studies have shown that the soil microbial community in alpine dry meadows of the Colorado Rocky Mountains changes in biomass, function, broad-level structure, and fungal diversity between winter and early summer. However, little specific information exists on the diversity of the alpine bacterial community or how it changes during this ecologically important period. We constructed clone libraries of 16S ribosomal DNA from alpine soil collected in winter, spring, and summer. We also cultivated bacteria from the alpine soil and measured the seasonal abundance of selected cultured isolates in hybridization experiments. The uncultured bacterial communities changed between seasons in diversity and abundance within taxa. TheAcidobacteriumdivision was most abundant in the spring. The winter community had the highest proportion ofActinobacteriaand members of theCytophaga/Flexibacter/Bacteroides(CFB) division. The summer community had the highest proportion of theVerrucomicrobiumdivision and of β-Proteobacteria. As a whole,α -Proteobacteriawere equally abundant in all seasons, although seasonal changes may have occurred within this group. A number of sequences from currently uncultivated divisions were found, including two novel candidate divisions. The cultured isolates belonged to the α-, β-, and γ-Proteobacteria, theActinobacteria, and the CFB groups. The only uncultured sequences that were closely related to the isolates were from winter and spring libraries. Hybridization experiments showed that actinobacterial and β-proteobacterial isolates were most abundant during winter, while the α- andγ -proteobacterial isolates tested did not vary significantly. While the cultures and clone libraries produced generally distinct groups of organisms, the two approaches gave consistent accounts of seasonal changes in microbial diversity.
Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule
Authors: Yoshino Tomoko , Takahashi Masayoshi , Takeyama Haruko , Okamura Yoshiko , Kato Fukuichi , Matsunaga Tadashi
Published: May 2004

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium
Magnetospirillum magneticum
AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.
New Method of Denitrification Analysis of
Bradyrhizobium
Field Isolates by Gas Chromatographic Determination of
15
N-Labeled N
2
Authors: Sameshima-Saito Reiko , Chiba Kaori , Minamisawa Kiwamu
Published: May 2004

To evaluate the denitrification abilities of many
Bradyrhizobium
field isolates, we developed a new
15
N-labeled N
2
detection methodology, which is free from interference from atmospheric N
2
contamination.
30
N
2
(
15
N
15
N) and
29
N
2
(
15
N
14
N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N
2
gas having natural abundance of
15
N (0.366 atom%) as a carrier gas. The detection limit was 0.04%
30
N
2
, and the linearity extended at least to 40%
30
N
2
. When
Bradyrhizobium japonicum
USDA110 was grown in cultures anaerobically with
15
NO
3
−
, denitrification product (
30
N
2
) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites,
Bradyrhizobium
species, and the
hup
genotype.