bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of Tn
5096
-Based Transposon Mutagenesis in
Gordonia polyisoprenivorans
Authors: Banh Quyen , Arenskötter Matthias , Steinbüchel Alexander
Published: September 2005

The transposons Tn
5
, Tn
10
, Tn
611
, and Tn
5096
were characterized regarding transposition in
Gordonia polyisoprenivorans
strain VH2. No insertional mutants were obtained employing Tn
5
or Tn
10
. The thermosensitive plasmid pCG79 harboring Tn
611
integrated into the chromosome of
G. polyisoprenivorans
; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn
5096
-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn
5096
as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(
cis
-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(
cis
-1,4-isoprene). One rubber-negative mutant was disrupted in
mcr
, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(
cis
-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.
Isolation of a Hop-Sensitive Variant ofLactobacillus lindneriand Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria
Authors: Suzuki Koji , Iijima Kazumaru , Ozaki Kazutaka , Yamashita Hiroshi
Published: September 2005

We have isolated a hop-sensitive variant of the beer spoilage bacteriumLactobacillus lindneriDSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designatedhorBLandhorCLthat encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs ofhorBandhorC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those ofhorAand ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use ofhorB/horCandhorAis able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison ofhorBandhorChomologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution ofhorBandhorChomologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.
1
H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5-
f
]Quinoline by Human Intestinal Microbiota
Authors: Humblot Christèle , Combourieu Bruno , Väisänen Marja-Liisa , Furet Jean-Pierre , Delort Anne-Marie , Rabot Sylvie
Published: September 2005

2-Amino-3-methylimidazo[4,5-
f
]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 μM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ
1
H nuclear magnetic resonance (
1
H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and
1
H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction:
Bacteroides thetaiotaomicron
(
n
= 2),
Clostridium clostridiiforme
(
n
= 3),
Clostridium perfringens
(
n
= 1), and
Escherichia coli
(
n
= 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.
Improvement of the Fatty Acid Composition of an Oil-Producing Filamentous Fungus,
Mortierella alpina
1S-4, through RNA Interference with Δ12-Desaturase Gene Expression
Authors: Takeno Seiki , Sakuradani Eiji , Tomi Akiko , Inohara-Ochiai Misa , Kawashima Hiroshi , Ashikari Toshihiko , Shimizu Sakayu
Published: September 2005

An oleaginous fungus,
Mortierella alpina
1S-4, is used commercially for arachidonic acid production. Δ12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in
M. alpina
1S-4, we silenced the Δ12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Δ12-desaturation-defective mutants previously identified. Thus, RNAi occurs in
M. alpina
and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.
New Chitosan-Degrading Strains That Produce Chitosanases Similar to ChoA of
Mitsuaria chitosanitabida
Authors: Yun ChoongSoo , Amakata Daiki , Matsuo Yasuhiro , Matsuda Hideyuki , Kawamukai Makoto
Published: September 2005

The betaproteobacterium
Mitsuaria chitosanitabida
(formerly
Matsuebacter chitosanotabidus
) 3001 produces a chitosanase (ChoA) that is classified in glycosyl hydrolase family 80. While many chitosanase genes have been isolated from various bacteria to date, they show limited homology to the
M. chitosanitabida
3001 chitosanase gene (
choA
). To investigate the phylogenetic distribution of chitosanases analogous to ChoA in nature, we identified 67 chitosan-degrading strains by screening and investigated their physiological and biological characteristics. We then searched for similarities to ChoA by Western blotting and Southern hybridization and selected 11 strains whose chitosanases showed the most similarity to ChoA. PCR amplification and sequencing of the chitosanase genes from these strains revealed high deduced amino acid sequence similarities to ChoA ranging from 77% to 99%. Analysis of the 16S rRNA gene sequences of the 11 selected strains indicated that they are widely distributed in the β and γ subclasses of
Proteobacteria
and the
Flavobacterium
group. These observations suggest that the ChoA-like chitosanases that belong to family 80 occur widely in a broad variety of bacteria.
Colonization of Cattle Intestines byCampylobacter jejuniandCampylobacter lanienae
Authors: Inglis G. Douglas , Kalischuk Lisa D. , Busz Hilma W. , Kastelic John P.
Published: September 2005

The location and abundance ofCampylobacter jejuniandCampylobacter lanienaein the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shedC. jejuniandC. lanienae(steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed.CampylobacterDNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. AlthoughC. jejuniandC. lanienaeDNA were detected in both animals, only steer A contained substantial quantities ofC. jejuniDNA. In both digesta and tissues of steer A,C. jejuniwas present in the duodenum and jejunum. Considerable quantities ofC. jejuniDNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities ofC. lanienaeDNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associatedC. lanienaeDNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance ofC. jejuniandC. lanienaeDNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA ofC. jejuniandC. lanienaewere detected in the digesta of 57% and 95% of the animals, respectively.C. jejuniassociated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance ofC. jejuniDNA in the ileum and rectum.C. lanienaewas most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore,C. jejunicolonized the proximal small intestine of asymptomatic beef cattle, whereasC. lanienaeprimarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.
Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression
Authors: He Zhili , Wu Liyou , Fields Matthew W. , Zhou Jizhong
Published: September 2005

Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a whole-genome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from
Shewanella oneidensis
MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. Hybridizations conducted at 42°C, 45°C, 50°C, and 60°C indicated that good sensitivities were obtained at 45°C for oligonucleotide probes in the presence of 50% formamide, under which conditions specific signals were detected by both PCR and oligonucleotide probes. Signal intensity increased as the length of the oligonucleotide probe increased, and the 70-mer oligonucleotide probes produced signal intensities similar to the intensities obtained with the PCR probes and detected numbers of open reading frames similar to the numbers detected with the PCR probes. PCR amplicon, 70-mer, 60-mer, and 50-mer arrays had detection sensitivities of 5.0, 25, 100, and 100 ng of genomic DNA, which were equivalent to approximately 1.9 × 10
6
, 9.2 × 10
6
, 3.7 × 10
7
, and 3.7 × 10
7
copies, respectively, when the array was hybridized with genomic DNA. To evaluate differential gene expression under experimental conditions,
S. oneidensis
MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.
Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in
Staphylococcus haemolyticus
Grown in Toluene
Authors: Nielsen Lindsey E. , Kadavy Dana R. , Rajagopal Soumitra , Drijber Rhae , Nickerson Kenneth W.
Published: September 2005

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of
Staphylococcus haemolyticus
which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and
p
-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus,
S. haemolyticus
alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in
S. haemolyticus
is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of
Staphylococcus aureus
and five strains of
Staphylococcus epidermidis
, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.
The Microcystin Composition of the Cyanobacterium
Planktothrix agardhii
Changes toward a More Toxic Variant with Increasing Light Intensity
Authors: Tonk Linda , Visser Petra M. , Christiansen Guntram , Dittmann Elke , Snelder Eveline O. F. M. , Wiedner Claudia , Mur Luuc R. , Huisman Jef
Published: September 2005

The cyanobacterium
Planktothrix agardhii
, which is dominant in many shallow eutrophic lakes, can produce hepatotoxic microcystins. Currently, more than 70 different microcystin variants have been described, which differ in toxicity. In this study, the effect of photon irradiance on the production of different microcystin variants by
P. agardhii
was investigated using light-limited turbidostats. Both the amount of the mRNA transcript of the
mcyA
gene and the total microcystin production rate increased with photon irradiance up to 60 μmol m
−2
s
−1
, but they started to decrease with irradiance greater than 100 μmol m
−2
s
−1
. The cellular content of total microcystin remained constant, independent of the irradiance. However, of the two main microcystin variants detected in
P. agardhii
, the microcystin-DeRR content decreased twofold with increased photon irradiance, whereas the microcystin-DeLR content increased threefold. Since microcystin-DeLR is considerably more toxic than microcystin-DeRR, this implies that
P. agardhii
becomes more toxic at high light intensities.
13
C-Carrier DNA Shortens the Incubation Time Needed To Detect Benzoate-Utilizing Denitrifying Bacteria by Stable-Isotope Probing
Authors: Gallagher E. , McGuinness L. , Phelps C. , Young L. Y. , Kerkhof L. J.
Published: September 2005

The active bacterial community able to utilize benzoate under denitrifying conditions was elucidated in two coastal sediments using stable-isotope probing (SIP) and
nosZ
gene amplification. The SIP method employed samples from Norfolk Harbor, Virginia, and a Long-Term Ecosystem Observatory (no. 15) off the coast of Tuckerton, New Jersey. The SIP method was modified by use of archaeal carrier DNA in the density gradient separation. The carrier DNA significantly reduced the incubation time necessary to detect the
13
C-labeled bacterial DNA from weeks to hours in the coastal enrichments. No denitrifier DNA was found to contaminate the archaeal
13
C-carrier when [
12
C]benzoate was used as a substrate in the sediment enrichments. Shifts in the activity of the benzoate-utilizing denitrifying population could be detected throughout a 21-day incubation. These results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader(s) in a bacterial population and to document the cross-feeding microbial community.
Quorum-Sensing Mutations Affect Attachment and Stability of
Burkholderia cenocepacia
Biofilms
Authors: Tomlin Kerry L. , Malott Rebecca J. , Ramage Gordon , Storey Douglas G. , Sokol Pamela A. , Ceri H.
Published: September 2005

Biofilm formation in
Burkholderia cenocepacia
has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the
cepIR
and
cciIR
quorum-sensing systems of
Burkholderia cenocepacia
K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the
cepI
and
cepR
mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the
cciI
mutant nor the double
cciI cepI
mutant was deficient in attachment, and restoration of the
cciI
gene resulted in less attachment than for the mutants. Meanwhile, the
cciR
mutant did show a significant reduction in attachment, as did the
cciR cepIR
mutant. While there was no change in antibiotic susceptibility with the individual
cepIR
and
cciIR
mutants, the
cepI cciI
mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the
cepI
and
cepR
mutants. Flow cell analysis of the individual
cepIR
mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of
B. cenocepacia
that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.
New Gammaproteobacteria Associated with Blood-Feeding Leeches and a Broad Phylogenetic Analysis of Leech Endosymbionts
Authors: Perkins Susan L. , Budinoff Rebecca B. , Siddall Mark E.
Published: September 2005

Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech,
Haementeria ghilianii
, and two others from
Placobdelloides
species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class
Gammaproteobacteria
. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus
Placobdella
as well as
Aeromonas veronii
from the medicinal leech,
Hirudo medicinalis
, and a
Rickettsia
sp. detected in
Hemiclepsis marginata
. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.
Extensive Diversity of Ionizing-Radiation-Resistant Bacteria Recovered from Sonoran Desert Soil and Description of Nine New Species of the GenusDeinococcusObtained from a Single Soil Sample
Authors: Rainey Fred A. , Ray Keren , Ferreira Margarida , Gatz Bridget Z. , Nobre M. Fernanda , Bagaley Danielle , Rash Brian A. , Park Mie-Jung , Earl Ashlee M. , Shank Nicole C. , Small Alanna M. , Henk Margaret C. , Battista John R. , Kämpfer Peter , da Costa Milton S.
Published: September 2005

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a60Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the generaDeinococcus,Geodermatophilus, andHymenobacterwere still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60Deinococcusstrains, the characterization of which revealed nine novel species of this genus.
Identification of Genes Induced in
Listeria monocytogenes
during Growth and Attachment to Cut Cabbage, Using Differential Display
Authors: Palumbo Jeffrey D. , Kaneko Aya , Nguyen Kimanh D. , Gorski Lisa
Published: September 2005

The food-borne pathogen
Listeria monocytogenes
is a ubiquitous soil bacterium with the potential to contaminate fresh produce during cultivation and postharvest processing. In order to identify potential mechanisms by which
L. monocytogenes
may successfully attach to and colonize fresh produce, gene expression in
L. monocytogenes
cells inoculated onto fresh-cut cabbage was compared to gene expression in cells grown under control conditions. Differential display of reverse transcriptase PCR fragments amplified with a set of 81 arbitrary primers allowed the isolation and identification of 32
L. monocytogenes
gene fragments that were observed to be more highly expressed under cabbage-associated conditions. Genes involved in carbohydrate, amino acid, and nucleic acid metabolism, motility and cell division, and transport were identified, as were a number of open reading frames (ORFs) encoding putative proteins with no known functions. Site-directed mutations in two ORFs encoding potential cell surface-associated proteins and a third ORF encoding a putative regulatory protein had no effect on the mutants' capacity to attach to fresh-cut cabbage. Although this study did not show clearly the impact of the differentially expressed genes on growth on cabbage, it is a first step in identifying some of the genetic factors that are potentially involved.
Impact of Phages on Two-Species Bacterial Communities
Authors: Harcombe W. R. , Bull J. J.
Published: September 2005

A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages,Escherichia coliB andSalmonella entericaserovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of twoE. coli-specific phages (T7 or T5) was added to the mixed culture,E. colibecame extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though theE. colidensities with phage reached high levels whenSalmonellawas absent. In contrast, the addition of a phage that attacked onlySalmonella(SP6) led to transient decreases in the bacterial number whetherE. coliwas absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria.
Inhibition of NO
3
−
and NO
2
−
Reduction by Microbial Fe(III) Reduction: Evidence of a Reaction between NO
2
−
and Cell Surface-Bound Fe
2+
Authors: Coby Aaron J. , Picardal Flynn W.
Published: September 2005

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO
3
−
and NO
2
−
(NO
x
−
) reduction by
Shewanella putrefaciens
200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe
2+
and NO
2
−
. This coating could then inhibit reduction of NO
x
−
by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of ∼3 mM NO
3
−
reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe
2+
and NO
2
−
, demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe
2+
onto
Paracoccus denitrificans
inhibited NO
x
−
reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO
2
−
was chemically reduced to N
2
O by goethite or cell-sorbed Fe
2+
, but not at appreciable rates by aqueous Fe
2+
. Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe
2+
and NO
2
−
. The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.
Resistance of Marine Bacterioneuston to Solar Radiation
Authors: Agogué Hélène , Joux Fabien , Obernosterer Ingrid , Lebaron Philippe
Published: September 2005

A total of 90 bacterial strains were isolated from the sea surface microlayer (i.e., bacterioneuston) and underlying waters (i.e., bacterioplankton) from two sites of the northwestern Mediterranean Sea. The strains were identified by sequence analysis, and growth recovery was investigated after exposure to simulated solar radiation. Bacterioneuston and bacterioplankton isolates were subjected to six different exposure times, ranging from 0.5 to 7 h of simulated noontime solar radiation. Following exposure, the growth of each isolate was monitored, and different classes of resistance were determined according to the growth pattern. Large interspecific differences among the 90 marine isolates were observed. Medium and highly resistant strains accounted for 41% and 22% of the isolates, respectively, and only 16% were sensitive strains. Resistance to solar radiation was equally distributed within the bacterioneuston and bacterioplankton. Relative contributions to the highly resistant class were 43% for γ-proteobacteria and 14% and 8% for α-proteobacteria and the
Cytophaga/Flavobacterium/Bacteroides
(CFB) group, respectively. Within the γ-proteobacteria, the
Pseudoalteromonas
and
Alteromonas
genera appeared to be highly resistant to solar radiation. The majority of the CFB group (76%) had medium resistance. Our study further provides evidence that pigmented bacteria are not more resistant to solar radiation than nonpigmented bacteria.
Survival of
Mycobacterium avium
subsp.
paratuberculosis
in Dam Water and Sediment
Authors: Whittington Richard J. , Marsh Ian B. , Reddacliff Leslie A.
Published: September 2005

In a previous longitudinal study,
Mycobacterium avium
subsp.
paratuberculosis
survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of
M. avium
subsp.
paratuberculosis
infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.
Enzyme-Coupled Assay for β-Xylosidase Hydrolysis of Natural Substrates
Authors: Wagschal Kurt , Franqui-Espiet Diana , Lee Charles C. , Robertson George H. , Wong Dominic W. S.
Published: September 2005

We describe here a new enzyme-coupled assay for the quantitation of
d
-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present
d
-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from
N
-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at ∼400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3′-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 β-xylosidase from
Thermoanaerobacterium
sp. strain JW/SL YS485 (Swiss-Prot accession no.
O30360
). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations
k
cat
was 2.7 ± 0.4 s
−1
,

K
m

was 3.3 ± 0.7 mM, and
k
cat
/

K
m

was 0.82 ± 0.21 mM
−1
 · s
−1
. Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose

K
i

was 1.7 ± 0.1 mM,
k
cat
was 2.0 ± 0.1 s
−1
,

K
m

was 0.144 ± 0.011 mM, and
k
cat
/

K
m

was 14 ± 1.3 mM
−1
 · s
−1
.
Overexpression of an Anti-CD3 Immunotoxin Increases Expression and Secretion of Molecular Chaperone BiP/Kar2p byPichia pastoris
Authors: Liu Yuan Yi , Woo Jung Hee , Neville David M.
Published: September 2005

We previously reported that the secretory capacity ofPichia pastorisis limited with respect to the secretion of a 96.5-kDa bivalent anti-CD3 immunotoxin; double-copy expression generated more translation products than single-copy expression but did not increase the secretion of the immunotoxin. InSaccharomyces cerevisiaeheterologous protein secretion has been reported to increase the expression of molecular chaperones, most prominently BiP/Kar2p. We therefore investigated the relationships between immunotoxin secretion and Kar2p expression inP. pastoris. We found that expression of the immunotoxin inP. pastorisincreased the expression of Kar2p to levels that surpassed the retrieval capacity of the cell, leading to secretion of Kar2p into the medium. The level of Kar2p secretion was correlated with the copy number of the immunotoxin gene. Intracellular Kar2p was found to bind exclusively to the unprocessed immunotoxin containing the prosequence of α-factor in the endoplasmic reticulum. These results show that Kar2p is intimately involved in immunotoxin secretion inP. pastoris. The limited capacity ofP. pastoristo retain a sufficiently high level of intracellular Kar2p may be a factor restricting the production of the immunotoxin.
Temporal Patterns of Nitrogenase Gene (
nifH
) Expression in the Oligotrophic North Pacific Ocean
Authors: Church Matthew J. , Short Cindy M. , Jenkins Bethany D. , Karl David M. , Zehr Jonathan P.
Published: September 2005

Dinitrogen (N
2
)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity. In this study, we examined the presence and expression of specific planktonic nitrogenase genes (
nifH
) in the upper ocean (0 to 175 m) at Station ALOHA in the oligotrophic North Pacific Ocean. Clone libraries constructed from reverse-transcribed PCR-amplified mRNA revealed six unique phylotypes. Five of the
nifH
phylotypes grouped with sequences from unicellular and filamentous cyanobacteria, and one of the phylotypes clustered with γ-proteobacteria. The cyanobacterial
nifH
phylotypes retrieved included two sequence types that phylogenetically grouped with unicellular cyanobacteria (termed groups A and B), several sequences closely related (97 to 99%) to
Trichodesmium
spp. and
Katagnymene spiralis
, and two previously unreported phylotypes clustering with heterocyst-forming
nifH
cyanobacteria. Temporal patterns of
nifH
expression were evaluated using reverse-transcribed quantitative PCR amplification of
nifH
gene transcripts. The filamentous and presumed unicellular group A cyanobacterial phylotypes exhibited elevated
nifH
transcription during the day, while members of the group B (closely related to
Crocosphaera watsonii
) unicellular phylotype displayed greater
nifH
transcription at night. In situ
nifH
expression by all of the cyanobacterial phylotypes exhibited pronounced diel periodicity. The γ-proteobacterial phylotype had low transcript abundance and did not exhibit a clear diurnal periodicity in
nifH
expression. The temporal separation of
nifH
expression by the various phylotypes suggests that open ocean diazotrophic cyanobacteria have unique in situ physiological responses to daily fluctuations of light in the upper ocean.
Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions
Authors: Kühn Inger , Iversen Aina , Finn Maria , Greko Christina , Burman Lars G. , Blanch Anicet R. , Vilanova Xavier , Manero Albert , Taylor Huw , Caplin Jonathan , Domínguez Lucas , Herrero Inmaculada A. , Moreno Miguel A. , Möllby Roland
Published: September 2005

Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE wereEnterococcus faeciumcarryingvanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.
Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome
Authors: Wilson S. C. , Wu C. , Andriychuk L. A. , Martin J. M. , Brasel T. L. , Jumper C. A. , Straus D. C.
Published: September 2005

The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of
Stachybotrys chartarum
,
Chaetomium globosum
,
Penicillium chrysogenum
, and
Cladosporium cladosporioides
were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h.
C. globosum
was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of
S. chartarum
were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for
C. globosum
colonies which were inactivated an average of 89%. More than 99% of ascospores of
C. globosum
were nonculturable. For all ascospore counts, mean test readings were lower than the controls (
P
< 0.001), indicating that some ascospores may also have been destroyed. Colonies of
S. chartarum
were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of
C. globosum
can play a slightly protective role for the ascospores and that
S. chartarum
, while affected by the fumigation treatment, still remains toxic.
Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms
Authors: Steinberger R. E. , Holden P. A.
Published: September 2005

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that
Pseudomonas aeruginosa
unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for
P. aeruginosa
biofilms. Significantly more eDNA was produced in
P. aeruginosa
and
Pseudomonas putida
biofilms than in
Rhodococcus erythropolis
or
Variovorax paradoxus
biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.
Dual Role of Response Regulator StyR in Styrene Catabolism Regulation
Authors: Leoni Livia , Rampioni Giordano , Di Stefano Valeria , Zennaro Elisabetta
Published: September 2005

In
Pseudomonas fluorescens
ST, the promoter of the styrene catabolic operon, P
styA
, is induced by styrene and repressed by the addition of preferred carbon sources. P
styA
is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in P
styA
regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on P
styA
. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the P
styA
promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of P
styA
. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.
Genetic Determinants of Volatile-Thiol Release bySaccharomyces cerevisiaeduring Wine Fermentation
Authors: Howell Kate S. , Klein Mathias , Swiegers Jan H. , Hayasaka Yoji , Elsey Gordon M. , Fleet Graham H. , Høj Peter B. , Pretorius Isak S. , de Barros Lopes Miguel A.
Published: September 2005

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation,Saccharomyces cerevisiaemediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.
Hydroxylation and Carboxylation—Two Crucial Steps of Anaerobic Benzene Degradation by
Dechloromonas
Strain RCB
Authors: Chakraborty Romy , Coates John D.
Published: September 2005

Benzene is a highly toxic industrial compound that is essential to the production of various chemicals, drugs, and fuel oils. Due to its toxicity and carcinogenicity, much recent attention has been focused on benzene biodegradation, especially in the absence of molecular oxygen. However, the mechanism by which anaerobic benzene biodegradation occurs is still unclear. This is because until the recent isolation of
Dechloromonas
strains JJ and RCB no organism that anaerobically degraded benzene was available with which to elucidate the pathway. Although many microorganisms use an initial fumarate addition reaction for hydrocarbon biodegradation, the large activation energy required argues against this mechanism for benzene. Other possible mechanisms include hydroxylation, carboxylation, biomethylation, or reduction of the benzene ring, but previous studies performed with undefined benzene-degrading cultures were unable to clearly distinguish which, if any, of these alternatives is used. Here we demonstrate that anaerobic nitrate-dependent benzene degradation by
Dechloromonas
strain RCB involves an initial hydroxylation, subsequent carboxylation, and loss of the hydroxyl group to form benzoate. These studies provide the first pure-culture evidence of the pathway of anaerobic benzene degradation. The outcome of these studies also suggests that all anaerobic benzene-degrading microorganisms, regardless of their terminal electron acceptor, may use this pathway.