bluuebunny/crossref_metadata_2025 huggingface dataset:
Statistical Quantification of Detachment Rates and Size Distributions of Cell Clumps from Wild-Type (PAO1) and Cell Signaling Mutant (JP1)
Pseudomonas aeruginosa
Biofilms
Authors: Wilson Suzanne , Hamilton Martin A. , Hamilton Gordon C. , Schumann Margo R. , Stoodley Paul
Published: October 2004

The detachment of cells from bacterial biofilms is an important, yet poorly understood and largely unquantified phenomenon. Detached cell clumps from medical devices may form microemboli and lead to metastasis, especially if they are resistant to host defenses and antibiotics. In manufacturing plants detached clumps entering a process stream decrease product quality. Two strains of
Pseudomonas aeruginosa
, a wild type (PAO1) and a cell signaling mutant (JP1), were studied to (i) quantify and model detachment patterns and (ii) determine the influence of cell signaling on detachment. We collected effluent from a biofilm flowthrough reactor and determined the size distribution for cell detachment events by microscopic examination and image analysis. The two strains were similar in terms of both biofilm structure and detachment patterns. Most of the detachment events were single-cell events; however, multiple-cell detachment events contributed a large fraction of the total detached cells. The rates at which events containing multiple cells detached from the biofilm were estimated by fitting a statistical model to the size distribution data. For events consisting of at least 1,000 cells, the estimated rates were 4.5 events mm
−2
min
−1
for PAO1 and 4.3 events mm
−2
min
−1
for JP1. These rates may be significant when they are scaled up to the total area of a real biofilm-contaminated medical device surface and to the hours or days of patient exposure.
Characterization of Plasmid pOR1 from
Ornithobacterium rhinotracheale
and Construction of a Shuttle Plasmid
Authors: Jansen Ruud , Chansiripornchai Niwat , Gaastra Wim , van Putten Jos P. M.
Published: October 2004

The bacterium
Ornithobacterium rhinotracheale
has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of
O. rhinotracheale
. The system is based on a cryptic plasmid, pOR1, that was derived from an
O. rhinotracheale
strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several
rep
genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in
Actinobacillus actinomycetemcomitans
and a number of genes coding for proteins with unknown function. An
Escherichia coli
-
O. rhinotracheale
shuttle plasmid (pOREC1) was constructed by cloning the replication origin and
rep
genes from pOR1 and the
cfxA
gene from
Bacteroides vulgatus
, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using
E. coli
as a host. pOREC1 was electroporated into
O. rhinotracheale
and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into
E. coli
, demonstrating that pOREC1 acts as an independent replicon in both
E. coli
and
O. rhinotracheale,
fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.
Inheritance of Resistance to
Bacillus thuringiensis
subsp.
kurstaki
in
Trichoplusia ni
Authors: Janmaat Alida F. , Wang Ping , Kain Wendy , Zhao Jian-Zhou , Myers Judith
Published: October 2004

The genetic inheritance of resistance to a commercial formulation of
Bacillus thuringiensis
subsp.
kurstaki
was examined in a
Trichoplusia ni
colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F
1
reciprocal crosses and backcrosses between F
1
larvae and resistant (P
R
) and susceptible (P
S
) populations were assayed at different
B. thuringiensis
subsp.
kurstaki
concentrations. The responses of progeny of reciprocal F
1
crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F
1
larvae was slightly higher than that for P
S
, suggesting that resistance is partially recessive. The responses of both backcross progeny (F
1
× P
R
, F
1
× P
S
) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F
1
× P
R
backcross but decreased the correspondence with the F
1
× P
S
backcross results. This finding suggests that resistance to
B. thuringiensis
subsp.
kurstaki
in this
T. ni
population is due to more than one gene.
Chthoniobacter flavus
gen. nov., sp. nov., the First Pure-Culture Representative of Subdivision Two,
Spartobacteria
classis nov., of the Phylum
Verrucomicrobia
Authors: Sangwan Parveen , Chen Xiaolei , Hugenholtz Philip , Janssen Peter H.
Published: October 2004

The phylum
Verrucomicrobia
is increasingly recognized as an environmentally significant group of bacteria, particularly in soil habitats. At least six subdivisions of the
Verrucomicrobia
are resolved by comparative analysis of 16S rRNA genes, mostly obtained directly from environmental samples. To date, only two of these subdivisions (1 and 4) have characterized pure-culture representatives. We have isolated and characterized the first known pure-culture representative of subdivision 2. Strain Ellin428 is an aerobic heterotrophic bacterium that is able to grow with many of the saccharide components of plant biomass but does not grow with amino acids or organic acids other than pyruvate. Cells are yellow, rod-shaped, nonmotile, and gram-stain negative, and they contain peptidoglycan with direct cross-linkages of the A1γ
meso
-Dpm type. The isolate grows well at 25°C on a variety of standard biological media, including some used in the routine cultivation of bacteria from soil. The pH range for growth is 4.0 to 7.0. Low levels of menaquinones MK-10 and MK-11 were detected. The major cellular fatty acids are C
14:0
, a-C
15:0
, C
16:1ω7c
, and/or 2OH i-C
15:0
, and C
16:0
. The G+C content of the genomic DNA is 61 mol%. We propose a new genus and species,
Chthoniobacter flavus
gen. nov., sp. nov., with isolate Ellin428 as the type strain, and a new class for the subdivision to which it belongs,
Spartobacteria
classis nov. Environmental sequences indicate that the class
Spartobacteria
is largely represented by globally distributed, abundant, and active soil bacteria.
Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in
Sinorhizobium meliloti
Increases Its Ability To Nodulate Alfalfa
Authors: Ma Wenbo , Charles Trevor C. , Glick Bernard R.
Published: October 2004

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of
Rhizobium leguminosarum
bv.
viciae
128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (
acdS
) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (
lrpL
), from
R. leguminosarum
bv.
viciae
128C53K were introduced into
Sinorhizobium meliloti
, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing
S. meliloti
strains showed 35 to 40% greater efficiency in nodulating
Medicago sativa
(alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing
S. meliloti
strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.
Pathogenic
Aeromonas hydrophila
Serogroup O:14 and O:81 Strains with an S Layer
Authors: Esteve Consuelo , Alcaide Elena , Canals Rocío , Merino Susana , Blasco Dolores , Figueras Maria José , Tomás Juan M.
Published: October 2004

Five autoagglutinating
Aeromonas hydrophila
isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10
4.61
to 10
7.11
), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of
A. hydrophila
TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81
A. hydrophila
strains seemed to be primarily different from those previously purified from strains
A. hydrophila
TF7 and
Aeromonas salmonicida
A450 on the basis of colony hybridizations with both the structural genes
vapA
and
ahsA
. This is the first report of the presence of an S layer in mesophilic
Aeromonas
strains not belonging to serogroup O:11.
New Method for Estimating Bacterial Cell Abundances in Natural Samples by Use of Sublimation
Authors: Glavin Daniel P. , Cleaves H. James , Schubert Michael , Aubrey Andrew , Bada Jeffrey L.
Published: October 2004

We have developed a new method based on the sublimation of adenine from
Escherichia coli
to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500°C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from ∼10
5
to 10
9
E. coli
cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.
Improved Stress Tolerance of GroESL-OverproducingLactococcus lactisand ProbioticLactobacillus paracaseiNFBC 338
Authors: Desmond C. , Fitzgerald G. F. , Stanton C. , Ross R. P.
Published: October 2004

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probioticLactobacillus paracaseiNFBC 338 was increased under heat adaptation conditions (52°C for 15 min). Subsequently, thegroESLoperon ofL. paracaseiNFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred intoLactococcus lactis(pGRO1) andL. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54°C) and lactobacilli (at 60°C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.
Bacterial Community Associated with Black Band Disease in Corals
Authors: Frias-Lopez Jorge , Klaus James S. , Bonheyo George T. , Fouke Bruce W.
Published: October 2004

Black band disease (BBD) is a virulent polymicrobial disease primarily affecting massive-framework-building species of scleractinian corals. While it has been well established that the BBD bacterial mat is dominated by a cyanobacterium, the quantitative composition of the BBD bacterial mat community has not described previously. Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the infectious bacterial community of the bacterial mat causing BBD. These analyses revealed that the bacterial composition of the BBD mat does not vary between different coral species but does vary when different species of cyanobacteria are dominant within the mat. On the basis of the results of a new method developed to identify organisms detected by T-RFLP analysis, our data show that besides the cyanobacterium, five species of the division
Firmicutes
, two species of the
Cytophaga-Flexibacter-Bacteroides
(CFB) group, and one species of δ-proteobacteria are also consistently abundant within the infectious mat. Of these dominant taxa, six were consistently detected in healthy corals. However, four of the six were found in much higher numbers in BBD mats than in healthy corals. One species of the CFB group and one species of
Firmicutes
were not always associated with the bacterial communities present in healthy corals. Of the eight dominant bacteria identified, two species were previously found in clone libraries obtained from BBD samples; however, these were not previously recognized as important. Furthermore, despite having been described as an important component of the pathogenetic mat, a
Beggiatoa
species was not detected in any of the samples analyzed. These results will permit the dominant BBD bacteria to be targeted for isolation and culturing experiments aimed at deciphering the disease etiology.
Genome-Wide Transposon Mutagenesis of
Borrelia burgdorferi
for Identification of Phenotypic Mutants
Authors: Stewart Philip E. , Hoff Jessica , Fischer Elizabeth , Krum Jonathan G. , Rosa Patricia A.
Published: October 2004

The spirochete
Borrelia burgdorferi
is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a
mariner
transposon to function in
B. burgdorferi
, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in
B. burgdorferi
and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a
mariner
transposon to function in
B. burgdorferi
should aid in identifying virulence factors and novel gene products related to spirochete physiology.
Unexpectedly Diverse
Mesorhizobium
Strains and
Rhizobium leguminosarum
Nodulate Native Legume Genera of New Zealand, while Introduced Legume Weeds Are Nodulated by
Bradyrhizobium
Species
Authors: Weir Bevan S. , Turner Susan J. , Silvester Warwick B. , Park Duck-Chul , Young John M.
Published: October 2004

The New Zealand native legume flora are represented by four genera,
Sophora
,
Carmichaelia
,
Clianthus
, and
Montigena
. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds:
Acacia
spp. (wattles),
Cytisus scoparius
(broom), and
Ulex europaeus
(gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA,
atpD
,
glnII
, and
recA
gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus
Mesorhizobium
, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across
Mesorhizobium
spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as
Rhizobium leguminosarum
. In contrast, all sequences from isolates from introduced weeds aligned with
Bradyrhizobium
species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.
Multiple-Antibiotic Resistance of
Enterococcus
spp. Isolated from Commercial Poultry Production Environments
Authors: Hayes Joshua R. , English Linda L. , Carr Lewis E. , Wagner David D. , Joseph Sam W.
Published: October 2004

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered,
Enterococcus faecalis
(53%) and
E. faecium
(31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of
E. faecalis
was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of
E. faecium
were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the
E. faecium
isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the
E. faecalis
population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.
Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial
Authors: Emerson Ray J. , Camesano Terri A.
Published: October 2004

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study,
Candida parapsilosis
(ATCC 90018) and
Pseudomonas aeruginosa
(ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between
C. parapsilosis
and both unmodified silicone rubber and
P. aeruginosa
biofilms. Using
C. parapsilosis
cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle. On
P. aeruginosa
biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that
C. parapsilosis
may adhere to both silicone rubber and
P. aeruginosa
biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.
Accelerated Biodegradation of Cement by Sulfur-Oxidizing Bacteria as a Bioassay for Evaluating Immobilization of Low-Level Radioactive Waste
Authors: Aviam Orli , Bar-Nes Gabi , Zeiri Yehuda , Sivan Alex
Published: October 2004

Disposal of low-level radioactive waste by immobilization in cement is being evaluated worldwide. The stability of cement in the environment may be impaired by sulfur-oxidizing bacteria that corrode the cement by producing sulfuric acid. Since this process is so slow that it is not possible to perform studies of the degradation kinetics and to test cement mixtures with increased durability, procedures that accelerate the biodegradation are required. Semicontinuous cultures of
Halothiobacillus neapolitanus
and
Thiomonas intermedia
containing thiosulfate as the sole energy source were employed to accelerate the biodegradation of cement samples. This resulted in a weight loss of up to 16% after 39 days, compared with a weight loss of 0.8% in noninoculated controls. Scanning electron microscopy of the degraded cement samples revealed deep cracks, which could be associated with the formation of low-density corrosion products in the interior of the cement. Accelerated biodegradation was also evident from the leaching rates of Ca
2+
and Si
2+
, the major constituents of the cement matrix, and Ca exhibited the highest rate (up to 20 times greater than the control rate) due to the reaction between free lime and the biogenic sulfuric acid. Leaching of Sr
2+
and Cs
+
, which were added to the cement to simulate immobilization of the corresponding radioisotopes, was also monitored. In contrast to the linear leaching kinetics of calcium, silicon, and strontium, the leaching pattern of cesium produced a saturation curve similar to the control curve. Presumably, the leaching of cesium is governed by the diffusion process, whereas the leaching kinetics of the other three ions seems to governed by dissolution of the cement.
Galactose Metabolism by
Streptococcus mutans
Authors: Abranches Jacqueline , Chen Yi-Ywan M. , Burne Robert A.
Published: October 2004

The
galK
gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in
Streptococcus mutans
UA159. The
galK
knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that
S. mutans
does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the
galK
mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase,
lacR
, was insertionally inactivated. A
galK
and
lacR
mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of
lacR
mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the
lacA
gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of
S. mutans
is not present in the
gal
,
lac
, or
msm
operons.
Reduction of
Escherichia coli
O157:H7 Populations in Cattle by Addition of Colicin E7-Producing
E. coli
to Feed
Authors: Schamberger Gerry P. , Phillips Ronald L. , Jacobs Jennifer L. , Diez-Gonzalez Francisco
Published: October 2004

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic
Escherichia coli
strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10
7
CFU of
E. coli
(a mixture of eight colicinogenic
E. coli
strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant
E. coli
O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic
E. coli
dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with
E. coli
O157:H7. The numbers of
E. coli
O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (
P
< 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log
10
CFU/g was observed, with a maximum decrease of 1.8 log
10
CFU/g at day 21 (overall statistical significance,
P
= 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (
P
< 0.04). These results indicated that the daily addition of 10
8
CFU of colicin E7-producing
E. coli
per gram of feed could reduce the fecal shedding of serotype O157:H7.
Influence of Apple Cultivars on Inactivation of Different Strains of
Escherichia coli
O157:H7 in Apple Cider by UV Irradiation
Authors: Basaran N. , Quintero-Ramos A. , Moake M. M. , Churey J. J. , Worobo R. W.
Published: October 2004

This study examined the effect of different apple cultivars upon the UV inactivation of
Escherichia coli
O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10
6
to 10
7
CFU of three strains of
E. coli
O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm
2
. Bacterial populations for treated and untreated samples were then enumerated by using nonselective media.
E. coli
O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (α ≤ 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (α ≤ 0.05) differences in at least two of the
E. coli
strains used. Comparison of log reductions among the
E. coli
strains to the cider parameters of °Brix, pH, and malic acid content failed to show any statistically significant relationship (
R
2
≥ 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of
E. coli
O157:H7 tested.
Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria
Authors: Charpentier Emmanuelle , Anton Ana I. , Barry Peter , Alfonso Berenice , Fang Yuan , Novick Richard P.
Published: October 2004

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile
Escherichia coli
-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an
E. coli
replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the
Staphylococcus aureus
chromosome. The staphylococcal cadmium-inducible P

cad

-cadC
and constitutive P

blaZ

promoters were designed and analyzed in transcriptional fusions to the staphylococcal β-lactamase
blaZ
, the
Vibrio fischeri luxAB
, and the
Aequorea victoria
green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and
cis-trans
effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.
Transcriptional Analysis of Biofilm Formation Processes in the Anaerobic, Hyperthermophilic BacteriumThermotoga maritima
Authors: Pysz Marybeth A. , Conners Shannon B. , Montero Clemente I. , Shockley Keith R. , Johnson Matthew R. , Ward Donald E. , Kelly Robert M.
Published: October 2004

Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in theT. maritimagenome. Among the previously annotated genes in theT. maritimagenome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e.,Pseudomonasspecies,Escherichia coli, andStaphylococcus epidermidis). Most notably,T. maritimabiofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessileT. maritimacommunities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.
Helix 4 Mutants of the
Bacillus thuringiensis
Insecticidal Toxin Cry1Aa Display Altered Pore-Forming Abilities
Authors: Vachon Vincent , Préfontaine Gabrielle , Rang Cécile , Coux Florence , Juteau Marc , Schwartz Jean-Louis , Brousseau Roland , Frutos Roger , Laprade Raynald , Masson Luke
Published: October 2004

The role played by α-helix 4 of the
Bacillus thuringiensis
toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to
Manduca sexta
larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to
N
-methyl-
d
-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that α-helix 4 plays a crucial role in the mechanism of pore formation.
Detection of Low Levels of
Listeria monocytogenes
Cells by Using a Fiber-Optic Immunosensor
Authors: Geng Tao , Morgan Mark T. , Bhunia Arun K.
Published: October 2004

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of
Listeria monocytogenes
cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture
Listeria
cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for
L. monocytogenes
and showed a significantly higher signal strength than for other
Listeria
species or other microorganisms, including
Escherichia coli
,
Enterococcus faecalis
,
Salmonella enterica
,
Lactobacillus plantarum
,
Carnobacterium gallinarum
,
Hafnia alvei
,
Corynebacterium glutamicum
,
Enterobacter aerogenes
,
Pseudomonas aeruginosa
, and
Serratia marcescens
, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 × 10
3
CFU/ml for a pure culture of
L. monocytogenes
grown at 37°C. When
L. monocytogenes
was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 × 10
4
or 2.8 × 10
7
CFU/ml, respectively. In less than 24 h, this method could detect
L. monocytogenes
in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered
Listeria
enrichment broth.
Quorum Quenching: Enzymatic Disruption ofN-Acylhomoserine Lactone-Mediated Bacterial Communication inBurkholderia thailandensis
Authors: Ulrich Ricky L.
Published: October 2004

Many species of gram-negative bacteria communicate by synthesizing, secreting, and responding toN-acylhomoserine lactones (AHLs), a mechanism termed quorum sensing. Several investigations have characterized numerous AHL-degrading enzymes (AiiA lactonases) encoded by environmental isolates ofBacillusspp. TheBurkholderia thailandensisquorum system is comprised of at least three AHL synthases (AHSs) and five transcriptional regulators belonging to the LuxIR class of proteins. Expression of theBacillus anthracis(Ames strain) AiiA lactonase inB. thailandensiscompletely abolished the accumulation ofN-decanoylhomoserine lactone (C10-HSL) andN-octanoylhomoserine lactone (C8-HSL), reducedN-hexanoylhomoserine lactone (C6-HSL) levels, altered both swarming and twitching motility, caused a significant increase in generation time, and affected carbon metabolism. In contrast, heterologous expression of theBacillus cereusstrain A24 AiiA lactonase inB. thailandensisreduced the concentrations of C6-HSL, C8-HSL, and C10-HSL to nondetectable levels; altered both swarming and twitching motility; and caused fluctuations in carbon utilization. Individual disruption of theB. thailandensisAHSs, specifically disruption of thebtaI1andbtaI3genes, which encode the proteins that direct the synthesis of C8-HSL and C6-HSL, respectively, caused the hyper-beta-hemolysis of sheep erythrocytes on blood agar plates. In contrast, AHL cleavage inB. thailandensisby theBacillusAiiA lactonases failed to enhance beta-hemolytic activity. The results of this study demonstrate that heterologous expression ofBacillussp. AiiA lactonases inB. thailandensisreduced AHL accumulation, affected both swarming and twitching motility, increased generation time, altered substrate utilization, and prevented the beta-hemolysis of sheep erythrocytes.
Persistence of
Streptococcus mutans
in Stationary-Phase Batch Cultures and Biofilms
Authors: Renye John A. , Piggot Patrick J. , Daneo-Moore Lolita , Buttaro Bettina A.
Published: October 2004

Streptococcus mutans
is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of
S. mutans
strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation;
Bac
Light staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 10
5
CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.
Stratified Growth in
Pseudomonas aeruginosa
Biofilms
Authors: Werner Erin , Roe Frank , Bugnicourt Amandine , Franklin Michael J. , Heydorn Arne , Molin Søren , Pitts Betsey , Stewart Philip S.
Published: October 2004

In this study, stratified patterns of protein synthesis and growth were demonstrated in
Pseudomonas aeruginosa
biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible
gfpmut2
gene encoding a stable GFP. The second construct carried a GFP derivative,
gfp
-AGA, encoding an unstable GFP under the control of the growth-rate-dependent
rrnBp
1
promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 μm wide in colony biofilms and 30 μm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 μm into the biofilm.
P. aeruginosa
was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature
P. aeruginosa
biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.
Spatiotemporal Distribution of Marine Magnetotactic Bacteria in a Seasonally Stratified Coastal Salt Pond
Authors: Simmons S. L. , Sievert S. M. , Frankel R. B. , Bazylinski D. A. , Edwards K. J.
Published: October 2004

The occurrence and distribution of magnetotactic bacteria (MB) were studied as a function of the physical and chemical conditions in meromictic Salt Pond, Falmouth, Mass., throughout summer 2002. Three dominant MB morphotypes were observed to occur within the chemocline. Small microaerophilic magnetite-producing cocci were present at the top of the chemocline, while a greigite-producing packet-forming bacterium occurred at the base of the chemocline. The distributions of these groups displayed sharp changes in abundance over small length scales within the water column as well as strong seasonal fluctuations in population abundance. We identified a novel, greigite-producing rod in the sulfidic hypolimnion that was present in relatively constant abundance over the course of the season. This rod is the first MB that appears to belong to the γ-
Proteobacteria
, which may suggest an iron- rather than sulfur-based respiratory metabolism. Its distribution and phylogenetic identity suggest that an alternative model for the ecological and physiological role of magnetotaxis is needed for greigite-producing MB.
Comparative Analysis of Physical Maps of FourBacillus subtilis(natto) Genomes
Authors: Qiu Dongru , Fujita Kyoko , Sakuma Yuko , Tanaka Teruo , Ohashi Yoshiaki , Ohshima Hideyuki , Tomita Masaru , Itaya Mitsuhiro
Published: October 2004

The complete SfiI and I-CeuI physical maps of fourBacillus subtilis(natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of theB. subtilisstrain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SPβ,skinelement, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.
Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G
Authors: Hsu Jyh-Shing , Yang Yunn-Bor , Deng Chan-Hui , Wei Chia-Li , Liaw Shwu-Huey , Tsai Ying-Chieh
Published: October 2004

Deacetoxycephalosporin C synthase (expandase) from
Streptomyces clavuligerus
, encoded by
cefE
, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight
cefE
-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an
Escherichia coli
ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the
k
cat
/

K
m

ratio compared to the
S. clavuligerus
expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.
Abundance of Reverse Tricarboxylic Acid Cycle Genes in Free-Living Microorganisms at Deep-Sea Hydrothermal Vents
Authors: Campbell Barbara J. , Cary S. Craig
Published: October 2004

Since the discovery of hydrothermal vents more than 25 years ago, the Calvin-Bassham-Benson (Calvin) cycle has been considered the principal carbon fixation pathway in this microbe-based ecosystem. However, on the basis of recent molecular data of cultured free-living and noncultured episymbiotic members of the epsilon subdivision of
Proteobacteria
and earlier carbon isotope data of primary consumers, an alternative autotrophic pathway may predominate. Here, genetic and culture-based approaches demonstrated the abundance of reverse tricarboxylic acid cycle genes compared to the abundance of Calvin cycle genes in microbial communities from two geographically distinct deep-sea hydrothermal vents. PCR with degenerate primers for three key genes in the reverse tricarboxylic acid cycle and form I and form II of ribulose 1,5-bisphosphate carboxylase/oxygenase (Calvin cycle marker gene) were utilized to demonstrate the abundance of the reverse tricarboxylic acid cycle genes in diverse vent samples. These genes were also expressed in at least one chimney sample. Diversity, similarity matrix, and phylogenetic analyses of cloned samples and amplified gene products from autotrophic enrichment cultures suggest that the majority of autotrophs that utilize the reverse tricarboxylic acid cycle are members of the epsilon subdivision of
Proteobacteria
. These results parallel the results of previously published molecular surveys of 16S rRNA genes, demonstrating the dominance of members of the epsilon subdivision of
Proteobacteria
in free-living hydrothermal vent communities. Members of the epsilon subdivision of
Proteobacteria
are also ubiquitous in many other microaerophilic to anaerobic sulfidic environments, such as the deep subsurface. Therefore, the reverse tricarboxylic acid cycle may be a major autotrophic pathway in these environments and significantly contribute to global autotrophic processes.