bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of an ABC Transporter Gene,
mcyH
, Results in Loss of Microcystin Production in the Cyanobacterium
Microcystis aeruginosa
PCC 7806
Authors: Pearson Leanne A. , Hisbergues Michael , Börner Thomas , Dittmann Elke , Neilan Brett A.
Published: November 2004

The cyanobacterium
Microcystis aeruginosa
is widely known for its production of the potent hepatotoxin microcystin. Microcystin is synthesized nonribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55-kb microcystin synthetase (
mcy
) gene cluster. Also encoded within the
mcy
gene cluster is a putative ATP binding cassette (ABC) transporter, McyH. This study details the bioinformatic and mutational analyses of McyH and offers functional predictions for the hypothetical protein. The transporter is putatively comprised of two homodimers, each with an N-terminal hydrophobic domain and a C-terminal ATPase. Phylogenetically, McyH was found to cluster with members of the ABC-A
1
subgroup of ABC ATPases, suggesting an export function for the protein. Two
mcyH
null mutant (Δ
mcyH
) strains were constructed by partial deletion of the
mcyH
gene. Microcystin production was completely absent in these strains. While the
mcyH
deletion had no apparent effect on the transcription of other
mcy
genes, the complete microcystin biosynthesis enzyme complex could not be detected in Δ
mcyH
mutant strains. Finally, expression levels of McyH in the wild type and in Δ
mcyA
, Δ
mcyB
, and Δ
mcyH
mutants were investigated by using immunoblotting with an anti-McyH antibody. Expression of McyH was found to be reduced in Δ
mcyA
and Δ
mcyB
mutants and completely absent in the Δ
mcyH
mutant. By virtue of its association with the
mcy
gene cluster and the bioinformatic and experimental data presented in this study, we predict that McyH functions as a microcystin exporter and is, in addition, intimately associated with the microcystin biosynthesis pathway.
Fatty Acid Production from Amino Acids and α-Keto Acids by
Brevibacterium linens
BL2
Authors: Ganesan Balasubramanian , Seefeldt Kimberly , Weimer Bart C.
Published: November 2004

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of
Brevibacterium linens
BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of
B. linens
ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.
Air- and Dustborne Mycoflora in Houses Free of Water Damage and Fungal Growth
Authors: Horner W. Elliott , Worthan Anthony G. , Morey Philip R.
Published: November 2004

Typically, studies on indoor fungal growth in buildings focus on structures with known or suspected water damage, moisture, and/or indoor fungal growth problems. Reference information on types of culturable fungi and total fungal levels are generally not available for buildings without these problems. This study assessed 50 detached single-family homes in metropolitan Atlanta, Ga., to establish a baseline of “normal and typical” types and concentrations of airborne and dustborne fungi in urban homes which were predetermined not to have noteworthy moisture problems or indoor fungal growth. Each home was visually examined, and samples of indoor and outdoor air and of indoor settled dust were taken in winter and summer. The results showed that rankings by prevalence and abundance of the types of airborne and dustborne fungi did not differ from winter to summer, nor did these rankings differ when air samples taken indoors were compared with those taken outdoors. Water indicator fungi were essentially absent from both air and dust samples. The air and dust data sets were also examined specifically for the proportions of colonies from ecological groupings such as leaf surface fungi and soil fungi. In the analysis of dust for culturable fungal colonies, leaf surface fungi constituted a considerable portion (>20%) of the total colonies in at least 85% of the samples. Thus, replicate dust samples with less than 20% of colonies from leaf surface fungi are unlikely to be from buildings free of moisture or mold growth problems.
Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism
Authors: Conn Vanessa M. , Franco Christopher M. M.
Published: November 2004

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of
Microbispora
sp. strain EN2,
Streptomyces
sp. strain EN27, and
Nocardioides albus
EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.
Relationships of a Novel Lyme Disease Spirochete,
Borrelia spielmani
sp. nov., with Its Hosts in Central Europe
Authors: Richter Dania , Schlee Daniela B. , Allgöwer Rainer , Matuschka Franz-Rainer
Published: November 2004

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for
Borrelia afzelii
, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies,
Borrelia spielmani
sp. nov., one of several that cause Lyme disease in people.
Escherichia coli
Contamination of Vegetables Grown in Soils Fertilized with Noncomposted Bovine Manure: Garden-Scale Studies
Authors: Ingham Steven C. , Losinski Jill A. , Andrews Matthew P. , Breuer Jane E. , Breuer Jeffry R. , Wood Timothy M. , Wright Thomas H.
Published: November 2004

In this study we tested the validity of the National Organic Program (NOP) requirement for a ≥120-day interval between application of noncomposted manure and harvesting of vegetables grown in manure-fertilized soil. Noncomposted bovine manure was applied to 9.3-m
2
plots at three Wisconsin sites (loamy sand, silt loam, and silty clay loam) prior to spring and summer planting of carrots, radishes, and lettuce. Soil and washed (30 s under running tap water) vegetables were analyzed for indigenous
Escherichia coli
. Within 90 days, the level of
E. coli
in manure-fertilized soil generally decreased by about 3 log CFU/g from initial levels of 4.2 to 4.4 log CFU/g. Low levels of
E. coli
generally persisted in manure-fertilized soil for more than 100 days and were detected in enriched soil from all three sites 132 to 168 days after manure application. For carrots and lettuce, at least one enrichment-negative sample was obtained ≤100 days after manure application for 63 and 88% of the treatments, respectively. The current ≥120-day limit provided an even greater likelihood of not detecting
E. coli
on carrots (≥1 enrichment-negative result for 100% of the treatments). The rapid maturation of radishes prevented conclusive evaluation of a 100- or 120-day application-to-harvest interval. The absolute absence of
E. coli
from vegetables harvested from manure-fertilized Wisconsin soils may not be ensured solely by adherence to the NOP ≥120-day limit. Unless pathogens are far better at colonizing vegetables than indigenous
E. coli
strains are, it appears that the risk of contamination for vegetables grown in Wisconsin soils would be elevated only slightly by reducing the NOP requirement to ≥100 days.
Survival of
Shewanella oneidensis
MR-1 after UV Radiation Exposure
Authors: Qiu Xiaoyun , Sundin George W. , Chai Benli , Tiedje James M.
Published: November 2004

We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in
Shewanella oneidensis
MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among
Shewanella
strains examined, with D
37
and D
10
values of 5.6 and 16.5% of
Escherichia coli
K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m
−2
, 568 J of UVB m
−2
, 25 kJ of UVA m
−2
, and 558 J of solar UVB m
−2
, respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10
−5
to 10
−6
. Unlike in
E. coli
, the expression levels of the nucleotide excision repair (NER) component genes
uvrA
,
uvrB
, and
uvrD
were not damage inducible in MR-1. Complementation of
Pseudomonas aeruginosa
UA11079 (
uvrA
deficient) with
uvrA
of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents.
Use of Merocyanine 540 for Photodynamic Inactivation of
Staphylococcus aureus
Planktonic and Biofilm Cells
Authors: Lin Hsiao-Yin , Chen Chin-Tin , Huang Ching-Tsan
Published: November 2004

Photodynamic inactivation of
Staphylococcus aureus
planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more
S. aureus
cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm
2
or to 20 μg of MC 540 per ml and a light dose of 450 J/cm
2
. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of
S. aureus
is closely related to the excitation level of MC 540.
Stages of Infection during the Tripartite Interaction between
Xenorhabdus nematophila
, Its Nematode Vector, and Insect Hosts
Authors: Sicard Mathieu , Brugirard-Ricaud Karine , Pagès Sylvie , Lanois Anne , Boemare Noel E. , Brehélin Michel , Givaudan Alain
Published: November 2004

Bacteria of the genus
Xenorhabdus
are mutually associated with entomopathogenic nematodes of the genus
Steinernema
and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled
Xenorhabdus nematophila
strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in
Spodoptera littoralis
larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the
Spodoptera
midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting
Spodoptera littoralis
larvae with nematodes harboring GFP-labeled
Xenorhabdus
. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine.
Xenorhabdus
began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of
Spodoptera
larvae. Comparative infection assays conducted with another insect,
Locusta migratoria
, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for
X. nematophila
within insects.
High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors
Authors: Tarre Sheldon , Green Michal
Published: November 2004

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg · liter
−1
), a very high nitrification rate of 5.6 g of N oxidized · liter
−1
 · day
−1
was achieved. The specific nitrification rate (0.55 g of N · g of biomass
−1
 · day
−1
) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (
15
N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass · g of N oxidized
−1
. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus
Nitrosomonas
, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.
Identification of a Third
msa
Gene in
Renibacterium salmoninarum
and the Associated Virulence Phenotype
Authors: Rhodes Linda D. , Coady Alison M. , Deinhard Rebecca K.
Published: November 2004

Renibacterium salmoninarum
, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes,
msa1
and
msa2
. We found evidence for a third
msa
gene,
msa3
, which appears to be a duplication of
msa1
. Unlike
msa1
and
msa2
,
msa3
is not present in all isolates of
R. salmoninarum
. The presence of the
msa3
locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the
msa3
locus reduced median survival in juvenile chinook salmon (
Oncorhynchus tshawytscha
) by an average of 34% at doses of ≤10
5
cells per fish compared to isolates lacking the
msa3
locus. In contrast, no difference in survival was observed at the highest dose, 10
6
cells per fish. The phenotype associated with the
msa3
locus and its nonuniform distribution may contribute to observed differences in virulence among
R. salmoninarum
isolates.
Transport of the Harmful Bloom Alga
Aureococcus anophagefferens
by Oceangoing Ships and Coastal Boats
Authors: Doblin Martina A. , Popels Linda C. , Coyne Kathryn J. , Hutchins David A. , Cary S. Craig , Dobbs Fred C.
Published: November 2004

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species
Aureococcus anophagefferens
(brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of
Aureococcus
(>22 ppt) and the low salinity of the residual ballast water (∼2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute
Aureococcus
. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of
Aureococcus
.
Coupling of Functional Gene Diversity and Geochemical Data from Environmental Samples
Authors: Palumbo A. V. , Schryver J. C. , Fields M. W. , Bagwell C. E. , Zhou J.-Z. , Yan T. , Liu X. , Brandt C. C.
Published: November 2004

Genomic techniques commonly used for assessing distributions of microorganisms in the environment often produce small sample sizes. We investigated artificial neural networks for analyzing the distributions of nitrite reductase genes (nirSandnirK) and two sets of dissimilatory sulfite reductase genes (dsrAB1anddsrAB2) in small sample sets. Data reduction (to reduce the number of input parameters), cross-validation (to measure the generalization error), weight decay (to adjust model parameters to reduce generalization error), and importance analysis (to determine which variables had the most influence) were useful in developing and interpreting neural network models that could be used to infer relationships between geochemistry and gene distributions. A robust relationship was observed between geochemistry and the frequencies of genes that were not closely related to known dissimilatory sulfite reductase genes (dsrAB2). Uranium and sulfate appeared to be the most related to distribution of two groups of these unusualdsrAB-related genes. For the other three groups, the distributions appeared to be related to pH, nickel, nonpurgeable organic carbon, and total organic carbon. The models relating the geochemical parameters to the distributions of thenirS,nirK, anddsrAB1genes did not generalize as well as the models fordsrAB2. The data also illustrate the danger (generating a model that has a high generalization error) of not using a validation approach in evaluating the meaningfulness of the fit of linear or nonlinear models to such small sample sizes.
Bacterium-Based NO
2
−
Biosensor for Environmental Applications
Authors: Nielsen Michael , Larsen Lars Hauer , Jetten Mike S. M. , Revsbech Niels Peter
Published: November 2004

A sensitive NO
2
−
biosensor that is based on bacterial reduction of NO
2
−
to N
2
O and subsequent detection of the N
2
O by a built-in electrochemical N
2
O sensor was developed. Four different denitrifying organisms lacking NO
3
−
reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO
2
−
tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was
Stenotrophomonas nitritireducens
, which is an organism with a denitrifying pathway deficient in both NO
3
−
and N
2
O reductases. Alternatively
Alcaligenes faecalis
could be used when acetylene was added to inhibit its N
2
O reductase. The macroscale biosensors constructed exhibited a linear NO
2
−
response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO
2
−
, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO
2
−
, and interference was observed only with NH
2
OH, NO, N
2
O, and H
2
S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO
2
−
biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO
2
−
biosensor were constructed and used to measure NO
2
−
profiles in marine sediment.
Diversity and Antagonistic Potential of Bacteria Associated with Bryophytes from Nutrient-Poor Habitats of the Baltic Sea Coast
Authors: Opelt Katja , Berg Gabriele
Published: November 2004

Very little is known about the interaction of bryophytes with bacteria. Therefore, we analyzed bacteria associated with three bryophyte species,
Tortula ruralis
,
Aulacomnium palustre
, and
Sphagnum rubellum
, which represent typical moss species of three nutrient-poor plant communities at the southern Baltic Sea coast in Germany. By use of two cultivation-independent techniques, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis of the 16S ribosomal DNA, a high degree of moss specificity was found for associated bacterial communities. This specificity could be further evidenced by a cultivation-dependent approach for the following parameters: (i) plate counts of bacteria on R2A medium, (ii) proportion of antagonistic isolates, (iii) antagonistic activity as well as spectrum against pathogens, and (iv) diversity and richness of antagonistic isolates. The proportion of isolates with antagonistic activity against the pathogenic model fungus
Verticillium dahliae
was highest for
S. rubellum
(31%), followed by
A. palustre
(17%) and
T. ruralis
(5%). A high percentage (99%) of moss-associated antagonistic bacteria produced antifungal compounds. The high recovery of antagonistic isolates strongly suggests that bryophytes represent an ecological niche which harbors a diverse and hitherto largely uncharacterized microbial population with yet unknown and untapped potential biotechnological applications, e.g., for biological control of plant pathogens.
Display of Biologically Functional Insecticidal Toxin on the Surface of λ Phage
Authors: Vílchez Susana , Jacoby Juliette , Ellar David J.
Published: November 2004

The successful use of
Bacillus thuringiensis
insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.
Effects of the Metalloid Oxyanion Tellurite (TeO
3
2−
) on Growth Characteristics of the Phototrophic Bacterium
Rhodobacter capsulatus
Authors: Borghese Roberto , Borsetti Francesca , Foladori Paola , Ziglio Giuliano , Zannoni Davide
Published: November 2004

This work examines the effects of potassium tellurite (K
2
TeO
3
) on the cell viability of the facultative phototroph
Rhodobacter capsulatus
. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO
3
2−
) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te
0
crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph
R. capsulatus
might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.
Postprocessing In Vitro Digestion Challenge To Evaluate Survival of
Escherichia coli
O157:H7 in Fermented Dry Sausages
Authors: Naim Fadia , Messier Serge , Saucier Linda , Piette Gabriel
Published: November 2004

Fermented dry sausages, inoculated with
Escherichia coli
O157:H7 during batter preparation, were submitted to an in vitro digestion challenge to evaluate the extent to which passage through the human gastrointestinal tract could inactivate the pathogenic cells, previously stressed by the manufacturing process. The numbers of surviving
E. coli
O157:H7 cells remained constant after a 1-min exposure of the finely chopped sausage to synthetic saliva or during the following 120-min exposure to synthetic gastric juice at an initial pH of 2.0. However, significant (
P
≤ 0.05) growth of the pathogen (1.03 to 2.16 log
10
CFU/g) was observed in a subsequent 250-min exposure to a synthetic pancreatic juice at pH 8.0. In a different set of experiments, fractions from the gastric suspension were transferred into the synthetic pancreatic juice at 30-min intervals to mimic the dynamics of gastric emptying. Concurrently, the pH of the remaining gastric fluid was reduced to 3.0, 2.5, and 2.0 to simulate the gradual reacidification of the stomach contents after the initial buffering effect resulting from meal ingestion. Under these new conditions, pathogen growth during pancreatic challenge was observed for the first few fractions released from the stomach (90 min of exposure [pH 2.5]), but growth was no longer possible in the fractions submitted to the most severe gastric challenge (120 min of exposure [pH < 2.2]).
Dynamics of Multiple
lin
Gene Expression in
Sphingomonas paucimobilis
B90A in Response to Different Hexachlorocyclohexane Isomers
Authors: Suar Mrutyunjay , van der Meer Jan Roelof , Lawlor Kirsten , Holliger Christof , Lal Rup
Published: November 2004

Sphingomonas paucimobilis
B90A is able to degrade the α-, β-, γ-, and δ-isomers of hexachlorocyclohexane (HCH). It contains the genes
linA
,
linB
,
linC
,
linD
,
linE
, and
linR
, which have been implicated in HCH degradation. In this study, dynamic expression of the
lin
genes was measured in chemostat-grown
S. paucimobilis
B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH,
linA
,
linB
, and
linC
were all expressed constitutively. In contrast,
linD
and
linE
were induced with α-HCH (2 mg/liter) and γ-HCH (7 mg/liter). A sharp increase in mRNA levels for
linD
and
linE
was observed from 10 to 45 min after the addition of α- or γ-HCH. Induction of
linD
and
linE
was not detectable upon the addition of 0.7 mg of γ-HCH per liter, although the compound was degraded by the cells. The addition of β-HCH (5 mg/liter) or δ-HCH (20 mg/liter) did not lead to
linE
and
linD
induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of β- and δ-HCH proceeds by a different pathway than that of α- and γ-HCH.
Molecular Characterization of
Brevibacillus laterosporus
and Its Potential Use in Biological Control
Authors: de Oliveira Edmar Justo , Rabinovitch Leon , Monnerat Rose Gomes , Passos Liana Konovaloff Jannotti , Zahner Viviane
Published: November 2004

Thirty-three strains of
Brevibacillus laterosporus
, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk
Biomphalaria glabrata
. Among the targets tested,
Biomphalaria glabrata
demonstrated the highest degree of sensitivity to
B. laterosporus
, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron
Anthonomus grandis
were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron
Anticarsia gemmatalis
. In contrast, no mortality was recorded among test populations of
Tenebrio molitor
or
Spodoptera frugiperda
. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of
B. laterosporus
when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related
Brevibacillus
species, indicating that they represent markers that are specific for
B. laterosporus
, which may prove useful for the isolation and identification of new strains of this species.
Pore Size Dependence on Growth Temperature Is a Common Characteristic of the Major Outer Membrane Protein OprF in Psychrotrophic and Mesophilic
Pseudomonas
Species
Authors: Jaouen Thomas , Dé Emmanuelle , Chevalier Sylvie , Orange Nicole
Published: November 2004

Pseudomonas
species adapt well to hostile environments, which are often subjected to rapid variations. In these bacteria, the outer membrane plays an important role in the sensing of environmental conditions such as temperature. In previous studies, it has been shown that in the psychrotrophic strain
P. fluorescens
MF0, the major porin OprF changes its channel size according to the growth conditions and could affect outer membrane permeability. Studies of the channel-forming properties of OprFs from
P. putida
01G3 and
P. aeruginosa
PAO1 in planar lipid bilayers generated similar results. The presence of a cysteine- or proline-rich cluster in the central linker region is not essential for channel size modulations. These findings suggest that OprF could adopt two alternative conformations in the outer membrane and that folding is thermoregulated. In contrast, no difference according to growth temperature was observed for structurally different outer membrane proteins, such as OprE3 from the
Pseudomonas
OprD family of specific porins. Our results are consistent with the fact that the decrease in channel size observed at low growth temperature is a particular feature of the OprF porin in various psychrotrophic and mesophilic
Pseudomonas
species isolated from diverse ecological niches. The ability to reduce outer membrane permeability at low growth temperature could provide these bacteria with adaptive advantages.
Life History Implications of rRNA Gene Copy Number in
Escherichia coli
Authors: Stevenson Bradley S. , Schmidt Thomas M.
Published: November 2004

The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of
Escherichia coli
in which one or two of the seven rRNA operons (
rrnA
and/or
rrnB
) were deleted. The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures. In batch cultures, the decrease in relative fitness corresponded to the number of rRNA operons deleted, which could be accounted for completely by increased lag times and decreased growth rates. The magnitude of the deleterious effect varied with the environment in which fitness was measured: the negative consequences of rRNA operon deletions increased under culture conditions permitting more-rapid growth. The rRNA operon deletion strains were not more effective competitors under the regimen of constant, limited resources provided in chemostat cultures. Enhanced fitness in chemostat cultures would have suggested a simple tradeoff in which deletion strains grew faster (due to more efficient resource utilization) under resource limitation. The contributions of growth rate, lag time,

K
s

, and death rate to the fitness of each strain were verified through mathematical simulation of competition experiments. These data support the hypothesis that multiple rRNA operons are a component of bacterial life history and that they confer a selective advantage permitting microbes to respond quickly and grow rapidly in environments characterized by fluctuations in resource availability.
Novel Sulfonolipid in the Extremely Halophilic Bacterium
Salinibacter ruber
Authors: Corcelli Angela , Lattanzio Veronica M. T. , Mascolo Giuseppe , Babudri Francesco , Oren Aharon , Kates Morris
Published: November 2004

Salinibacter ruber
is an extremely halophilic bacterium, phylogenetically affiliated with the
Flavobacterium
/
Cytophaga
branch of the domain
Bacteria
. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of
S. ruber
shows a characteristic peak at
m/z
660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]
−
of
m/z
660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-
O
-(13′-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus
Cytophaga
and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol.
153
:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta
528
:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at
m/z
660 the lipid signature of
Salinibacter
. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of
Salinibacter
within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.
Use of Cell Culture-PCR Assay Based on Combination of A549 and BGMK Cell Lines and Molecular Identification as a Tool To Monitor Infectious Adenoviruses and Enteroviruses in River Water
Authors: Lee Cheonghoon , Lee Seung-Hoon , Han Euiri , Kim Sang-Jong
Published: November 2004

Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.
Benthic and Pelagic Viral Decay Experiments: a Model-Based Analysis and Its Applicability
Authors: Fischer Ulrike R. , Weisz Willy , Wieltschnig Claudia , Kirschner Alexander K. T. , Velimirov Branko
Published: November 2004

The viral decay in sediments, that is, the decrease in benthic viral concentration over time, was recorded after inhibiting the production of new viruses. Assuming that the viral abundance in an aquatic system remains constant and that viruses from lysed bacterial cells replace viruses lost by decay, the decay of viral particles can be used as a measure of viral production. Decay experiments showed that this approach is a useful tool to assess benthic viral production. However, the time course pattern of the decay experiments makes their interpretation difficult, regardless of whether viral decay is determined in the water column or in sediments. Different curve-fitting approaches (logarithmic function, power function, and linear regression) to describe the time course of decay experiments found in the literature are used in the present study and compared to a proposed “exponential decay” model based on the assumption that at any moment the decay is proportional to the amount of viruses present. Thus, an equation of the form
d
VA/
dt
= −
k
× VA leading to the time-integrated form VA

t

= VA
0
×

e
−k

×

t

was used, where
k
represents the viral decay rate (h
−1
), VA

t

is the viral abundance (viral particles ml
−1
) at time
t
(h), and VA
0
is the initial viral abundance (viral particles ml
−1
). This approach represents the best solution for an accurate curve fitting based on a mathematical model for a realistic description of viral decay occurring in aquatic systems. Decay rates ranged from 0.0282 to 0.0696 h
−1
(mean, 0.0464 h
−1
). Additionally, a mathematical model is presented that enables the quantification of the viral control of bacterial production. The viral impact on bacteria based on decay rates calculated from the different mathematical approaches varied widely within one and the same decay experiment. A comparison of the viral control of bacterial production in different aquatic environments is, therefore, improper when different mathematical formulas are used to interpret viral decay experiments.
Denaturing Gradient Gel Electrophoretic Analysis of Ammonia-Oxidizing Bacterial Community Structure in the Lower Seine River: Impact of Paris Wastewater Effluents
Authors: Cébron Aurélie , Coci Manuela , Garnier Josette , Laanbroek Hendrikus J.
Published: November 2004

The Seine River is strongly affected by the effluents from the Achères wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from upstream and/or allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community in the lower Seine River and to analyze the temporal and spatial functioning of the system at several different sampling dates. A major revelation was the stability of the patterns. The CTO primers used in this study (G. A. Kowalchuk, J. R. Stephen, W. D. Boer, J. I. Prosser, T. M. Embley, and J. W. Woldendorp, Appl. Environ. Microbiol.
63:
1489-1497, 1997) were shown not to be completely specific to AOB of the β subclass of
Proteobacteria
. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the β subclass of
Proteobacteria
. The majority of AOB (75 to 90%) present in the lower Seine river downstream of the effluent output belong to lineage 6a, represented by
Nitrosomonas oligotropha-
and
Nitrosomonas ureae
-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions, i.e., an increase in the amount of suspended matter, a low ammonia concentration, and high turnover of organic matter. The rest of the AOB population was represented in equal proportions by
Nitrosospira
- and
Nitrosococcus mobilis
-like species.
Bacterial Diversity and Function of Aerobic Granules Engineered in a Sequencing Batch Reactor for Phenol Degradation
Authors: Jiang He-Long , Tay Joo-Hwa , Maszenan Abdul Majid , Tay Stephen Tiong-Lee
Published: November 2004

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of β-
Proteobacteria
and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.