bluuebunny/crossref_metadata_2025 huggingface dataset:
Chitinase Genes in Lake Sediments of Ardley Island, Antarctica
Authors: Xiao Xiang , Yin Xuebin , Lin Jian , Sun Liguang , You Ziyong , Wang Peng , Wang Fengping
Published: December 2005

A sediment core spanning approximately 1,600 years was collected from a lake on Ardley Island, Antarctica. The sediment core had been greatly influenced by penguin guano. Using molecular methods, the chitinolytic bacterial community along the sediment core was studied over its entire length. Primers targeting conserved sequences of the catalytic domains of family 18 subgroup A chitinases detected group A chitinases from a wide taxonomic range of bacteria. Using quantitative competitive PCR (QC-PCR), chitinase gene copies in each 1-cm section of the whole sediment column were quantified. QC-PCR determination of the chitinase gene copies indicated significant correlation with phosphorus and total organic carbon concentration, suggesting a historical connection between chitinase gene copies and the amount of penguin guano input into the lake sediment. Most of the chitinase genes cloned from the historic sediment core were novel. Analysis of the chitinase gene diversity in selected sediment layers and in the fresh penguin deposits indicated frequent shifts in the chitinolytic bacterial community over time. Sequence analysis of the 16S rRNA genes of chitinolytic bacteria isolated from the lake sediment revealed that the isolates belonged to
Janthinobacterium
species,
Stenotrophomonas
species of γ-
Proteobacteria
,
Cytophaga
species of the
Cytophaga
-
Flexibacter
-
Bacteroides
group, and
Streptomyces
and
Norcardiopsis
species of
Actinobacteria
. Chitinase gene fragments were cloned and sequenced from these cultivated chitinolytic bacteria. The phylogeny of the chitinase genes obtained from the isolates did not correspond well to that of the isolates, suggesting acquisition via horizontal gene transfer.
Class 1 Integrons and Tetracycline Resistance Genes in
Alcaligenes
,
Arthrobacter
, and
Pseudomonas
spp. Isolated from Pigsties and Manured Soil
Authors: Agersø Yvonne , Sandvang Dorthe
Published: December 2005

The presence of tetracycline resistance (Tc
r
) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc
r
gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species
Escherichia coli
,
Enterobacter
spp.,
Arthrobacter
spp.,
Alcaligenes
spp.,
Pseudomonas
spp., and
Corynebacterium glutamicum.
The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc
r
genes
tet
(A),
tet
(B), and
tet
(C), and all (
n
= 72) gram-positive isolates were screened for
tet
(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained
tet
genes also contained in-1, except one gram-negative isolate from a pigsty that contained
tet
(B). All gram-positive isolates with in-1 also contained
tet
(33). No isolates contained more than one
tet
gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc
r
and class I integrons from soil isolates to
Escherichia coli
and/or
Pseudomonas putida
. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc
r
but also multidrug resistance (caused by the presence
tet
genes and in-1) in bacteria.
Determination of the Viability of
Aeromonas hydrophila
in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods
Authors: Pianetti Anna , Falcioni Tania , Bruscolini Francesca , Sabatini Luigia , Sisti Elivio , Papa Stefano
Published: December 2005

The presence of
Aeromonas
spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of
Aeromonas hydrophila
in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.
Effects of Common Forage Phenolic Acids on
Escherichia coli
O157:H7 Viability in Bovine Feces
Authors: Wells J. E. , Berry E. D. , Varel V. H.
Published: December 2005

Ruminant animals are carriers of
Escherichia coli
O157:H7, and the transmission of
E. coli
O157:H7 from cattle to the environment and to humans is a concern. It is unclear if diet can influence the survivability of
E. coli
O157:H7 in the gastrointestinal system or in feces in the environment. Feces from cattle fed bromegrass hay or corn silage diets were inoculated with
E. coli
O157:H7, and the survival of this pathogen was analyzed. When animals consumed bromegrass hay for <1 month, viable
E. coli
O157:H7 was not recovered after 28 days postinoculation, but when animals consumed the diet for >1 month,
E. coli
O157:H7 cells were recovered for >120 days. Viable
E. coli
O157:H7 cells in feces from animals fed corn silage were detected until day 45 and differed little with the time on the diet. To determine if forage phenolic acids affected the viability of
E. coli
O157:H7, feces from animals fed corn silage or cracked corn were amended with common forage phenolic acids. When 0.5%
trans
-cinnamic acid or 0.5%
para
-coumaric acid was added to feces from silage-fed animals, the
E. coli
O157:H7 death rate was increased significantly (17-fold and 23-fold, respectively) compared to that with no addition. In feces from animals fed cracked corn,
E. coli
O157:H7 death rates were increased significantly with the addition of 0.1% and 0.5%
trans
-cinnamic acid (7- and 13-fold), 0.1% and 0.5%
p
-coumaric acid (3- and 8-fold), and 0.5% ferulic acid (3-fold). These data suggest that phenolic acids common to forage plants can decrease viable counts of
E. coli
O157:H7 shed in feces.
Cometabolism of Trihalomethanes by
Nitrosomonas europaea
Authors: Wahman David G. , Katz Lynn E. , Speitel Gerald E.
Published: December 2005

The ammonia-oxidizing bacterium
Nitrosomonas europaea
(ATCC 19718) was shown to degrade low concentrations (50 to 800 μg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants (

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) increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH
3
-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (

T
c

), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.
Horizontal Transfer of Bacterial Symbionts: Heritability and Fitness Effects in a Novel Aphid Host
Authors: Russell Jacob A. , Moran Nancy A.
Published: December 2005

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the
Gammaproteobacteria
to infect and spread within a novel host species, the pea aphid,
Acyrthosiphon pisum
. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting
A. pisum
. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize
A. pisum
as a host. One of three novel symbionts failed to undergo efficient maternal transmission in
A. pisum
, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.
Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from
Sphingomonas
sp. Strain AO1
Authors: Sasaki Miho , Akahira Ayako , Oshiman Ko-ichi , Tsuchido Tetsuaki , Matsumura Yoshinobu
Published: December 2005

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation
16:
449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by
Sphingomonas
sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450
bisd
), ferredoxin (Fd
bisd
), and ferredoxin reductase (Red
bisd
). We demonstrated that P450
bisd
and Fd
bisd
are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd
bisd
revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450
bisd
, in the presence of Fd
bisd
, Red
bisd
, and NADH, was able to convert BPA. The

K
m

and
k
cat
values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min
−1
, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450
bisd
might exhibit strict specificity. Two BPA degradation products of the P450
bisd
system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.
Microarray-Based Detection and Typing of the Rhizobium Nodulation Gene
nodC
: Potential of DNA Arrays To Diagnose Biological Functions of Interest
Authors: Bontemps Cyril , Golfier Geoffroy , Gris-Liebe Carine , Carrere Sébastien , Talini Luc , Boivin-Masson Catherine
Published: December 2005

Environmental screening of bacteria for the presence of genes of interest is a challenging problem, due to the high variability of the nucleotide sequence of a given gene between species. Here, we tackle this general issue using a particularly well-suited model system that consists of the nodulation gene
nodC
, which is shared by phylogenetically distant rhizobia. 41mer and 50mer oligonucleotides featuring the nucleotide diversity of two highly conserved regions of the NodC protein were spotted on glass slides and cross hybridized with the radioactive-labeled target genomic DNA under low-stringency conditions. Statistical analysis of the hybridization patterns allowed the detection of known, as well as new,
nodC
sequences and classified the rhizobial strains accordingly. The microarray was successfully used to type the
nodC
gene directly from legume nodules, thus eliminating the need of cultivation of the endosymbiont. This approach could be extended to a panel of diagnostic genes and constitute a powerful tool for studying the distribution of genes of interest in the environment, as well as for bacteria identification.
Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea
Authors: Manske Ann K. , Glaeser Jens , Kuypers Marcel M. M. , Overmann Jörg
Published: December 2005

The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophylle(BChle) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter−1. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChle. Only traces of BChle(8 ng liter−1) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m−2s−1at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of theChlorobiaceaeand was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m−2s−1for photosynthetic14CO2fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.
Heterologous Production of Antimicrobial Peptides in
Propionibacterium freudenreichii
Authors: Brede Dag Anders , Faye Therese , Stierli Melanie Patricia , Dasen Gottfried , Theiler Anita , Nes Ingolf F. , Meile Leo , Holo Helge
Published: December 2005

Heterologous bacteriocin production in
Propionibacterium freudenreichii
is described. We developed an efficient system for DNA shuttling between
Escherichia coli
and
P. freudenreichii
using vector pAMT1. It is based on the
P. freudenreichii
rolling-circle replicating plasmid pLME108 and carries the
cml
(A)/
cmx
(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (
pctA
) into pAMT1 under the control of the constitutive promoter (P
4
) yielded bacteriocin in amounts equal to those of the wild-type producer
Propionibacterium thoenii
419. The
P. freudenreichii
clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The
pamA
gene from
P. thoenii
419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in
P. freudenreichii
, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the
pamA
signal peptide and PAMP were fused in frame. The promoter region of
pamA
was identified by fusion of putative promoter fragments to the coding sequence of the
pctA
gene. The P
4
and P
pamp
promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.
Sequential Reductive Dechlorination of
meta
-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different
Chloroflexi
Phylotypes
Authors: Fagervold Sonja K. , Watts Joy E. M. , May Harold D. , Sowers Kevin R.
Published: December 2005

Three species within a deeply branching cluster of the
Chloroflexi
are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the
Chloroflexi
including
Dehalococcoides
spp. and the
o
-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the
para-
flanked
meta
position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the
Dehalococcoides
group. This same species reductively dechlorinated the
para
- and
ortho-
flanked
meta
-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the
o
-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the
Chloroflexi
exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of
Chloroflexi
with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.
Diversity of Carotenoid Synthesis Gene Clusters from Environmental
Enterobacteriaceae
Strains
Authors: Sedkova Natalia , Tao Luan , Rouvière Pierre E. , Cheng Qiong
Published: December 2005

Eight
Enterobacteriaceae
strains that produce zeaxanthin and derivatives of this compound were isolated from a variety of environmental samples. Phylogenetic analysis showed that these strains grouped with different clusters of
Erwinia
type strains. Four strains representing the phylogenetic diversity were chosen for further characterization, which revealed their genetic diversity as well as their biochemical diversity. The carotenoid synthesis gene clusters cloned from the four strains had three different gene organizations. Two of the gene clusters, those from strains DC416 and DC260, had the classical organization
crtEXYIBZ
; the gene cluster from DC413 had the rare organization
crtE-idi-XYIBZ
; and the gene cluster from DC404 had the unique organization
crtE-idi-YIBZ
. Besides the diversity in genetic organization, these genes also exhibited considerable sequence diversity. On average, they exhibited 60 to 70% identity with each other, as well as with the corresponding genes of the
Pantoea
type strains. The four different clusters were individually expressed in
Escherichia coli
, and the two
idi
-containing clusters gave more than fivefold-higher carotenoid titers than the two clusters lacking
idi
. Expression of the
crtEYIB
genes with and without
idi
confirmed the effect of increasing carotenoid titer by the type II
idi
gene linked with the carotenoid synthesis gene clusters.
Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland
Authors: Metje Martina , Frenzel Peter
Published: December 2005

The effects of temperature on rates and pathways of CH
4
production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH
4
and CO
2
over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH
3
F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH
4
· g [dry weight]
−1
· day
−1
), but the activity was relatively high even at 4°C (0.25 μmol CH
4
· g [dry weight]
−1
· day
−1
). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order
Methanobacteriales
, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H
2
sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.
Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis inSaccharomyces cerevisiae
Authors: Leonard Effendi , Yan Yajun , Lim Kok Hong , Koffas Mattheos A. G.
Published: December 2005

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing inSaccharomyces cerevisiaea four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain whenp-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.
Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from X
anthomonas campestris
pv. vesicatoria
Authors: Basim Huseyin , Minsavage Gerald V. , Stall Robert E. , Wang Jaw-Fen , Shanker Savita , Jones Jeffrey B.
Published: December 2005

We characterized the copper resistance genes in strain XvP26 of
Xanthomonas campestris
pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in
Xanthomonas
and
Pseudomonas
strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in
Pseudomonas syringae
pv. tomato and
Escherichia coli
, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in
E. coli
and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in
Sinorhizobium meliloti
, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant
X. campestris
pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in
X. campestris
pv. vesicatoria.
Bleeding Sap and Old Wood Are the Two Main Sources of Contamination of Merging Organs of Vine Plants byXylophilus ampelinus, the Causal Agent of Bacterial Necrosis
Authors: Grall S. , Roulland C. , Guillaumès J. , Manceau C.
Published: December 2005

The spatial distribution of vine plants contaminated byXylophilus ampelinus, the agent responsible for bacterial necrosis, was studied over a 5-year period within two vineyards in the Cognac area. Both vineyards were planted withVitis viniferacv. Ugni blanc but were different in age and agronomic location. The emission ofX. ampelinusin contaminated bleeding sap was observed during vine sprouting. Contaminated bleeding sap is an important source of inoculum for external contamination due to the high susceptibility of young merging shoots to the pathogen.X. ampelinusemission by bleeding sap was not affected by the age of the plants or the location of the vineyards. However, its emission was irregular with time, and it varied between two fruit canes from individual plants and between plants as well as between years. Moreover, the two vineyards appeared to be entirely contaminated. Consequently, the behavior of the pathogen is not predictable. The distribution of the pathogen inside vine plant organs was analyzed through the four growing seasons. The old wood was contaminated throughout the year and constituted a stock inoculum for endophytic contamination of crude sap during the winter and the spring. Despite the fact that most of the young green shoots were contaminated in May,X.ampelinuswas not found in green shoots in June and September, refuting the hypothesis of an epiphytic life of the pathogen under natural conditions. Although all plants were entirely contaminated in both vineyards, symptoms were rare and were observed on different plants each year.
Molecular Characterization ofClostridium perfringensIsolates from Humans with Sporadic Diarrhea: Evidence for Transcriptional Regulation of the Beta2-Toxin-Encoding Gene
Authors: Harrison Ben , Raju Deepa , Garmory Helen S. , Brett Moira M. , Titball Richard W. , Sarker Mahfuzur R.
Published: December 2005

Clostridium perfringenstype A food poisoning is caused byC. perfringensisolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused byC. perfringensplasmidcpeisolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14cpe+C. perfringensfecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified ascpe+type A, and 12 of the 14cpe+isolates carry theircpegene on the plasmid and 2 carry it on the chromosome. Interestingly,cpb2is present in only 12 plasmidcpeisolates; 11 isolates carrycpeandcpb2on different plasmids, butcpeandcpb2are located on the same plasmid in one isolate.C. perfringensenterotoxin is produced by all 14cpe+SD isolates. However, only 10 of the 12cpe+/cpb2+SD isolates produced CPB2, with significant variation in amounts. The levels ofcpb2mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypicalcpb2was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression ofcpb2mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94cpb2, which overexpressedcpb2mRNA, restored CPB2 production in F4969 (acpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin inC. perfringens-associated SD.
Visualization and Detection of Infectious Coxsackievirus Replication Using a Combined Cell Culture-Molecular Beacon Assay
Authors: Wang Aijun , Salazar Andre M. , Yates Marylynn V. , Mulchandani Ashok , Chen Wilfred
Published: December 2005

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5′ noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.
Diversity and Structure of Bacterial Chemolithotrophic Communities in Pine Forest and Agroecosystem Soils
Authors: Tolli J. , King G. M.
Published: December 2005

Obligate lithotrophs (e.g., ammonia oxidizers) and facultative lithotrophs (e.g., CO and hydrogen oxidizers) collectively comprise a phylogenetically diverse functional group that contributes significantly to carbon and nitrogen cycles in soils and plays important roles in trace gas dynamics (e.g., carbon monoxide and nitrous and nitric oxides) that affect tropospheric chemistry and radiative forcing. In spite of their diverse physiologies, facultative and obligate lithotrophs typically possess the Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisCO). In an effort designed to understand the structure of lithotrophic communities in soil, genomic DNA extracts from surface (0 to 2 cm) and subsurface (5 to 7 cm) soils have been obtained from two sites in a Georgia agroecosystem (peanut and cotton plots) and an unmanaged pine stand (>50 years old). The extracts have been used in PCR amplifications of the
cbbL
gene for the rubisCO large subunit protein.
cbbL
PCR products were cloned, sequenced, and subjected to phylogenetic and statistical analyses. Numerous novel lineages affiliated with the form IC clade (one of four form I rubisCO clades), which is typified by facultative lithotrophs, comprised lithotrophic communities from all soils. One of the form IC clone sequences clustered with a form IC clade of ammonia-oxidizing
Nitrosospira
. Distinct assemblages were obtained from each of the sites and from surface and subsurface soils. The results suggest that lithotrophic populations respond differentially to plant type and land use, perhaps forming characteristic associations. The paucity of clone sequences attributed to ammonia-oxidizing bacteria indicates that even though ammonia oxidation occurs in the various soils, the relevant populations are small compared to those of facultative lithotrophs.
New Multiple-Deletion Method for the
Corynebacterium glutamicum
Genome, Using a Mutant
lox
Sequence
Authors: Suzuki Nobuaki , Nonaka Hiroshi , Tsuge Yota , Inui Masayuki , Yukawa Hideaki
Published: December 2005

Due to the difficulty of multiple deletions using the Cre/
loxP
system, a simple, markerless multiple-deletion method based on a Cre
/
mutant
lox
system combining a right-element (RE) mutant
lox
site with a left-element (LE) mutant
lox
site was employed for large-scale genome rearrangements in
Corynebacterium glutamicum
. Eight distinct genomic regions that had been identified previously by comparative analysis of
C. glutamicum
R and
C. glutamicum
13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant
lox
sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant
lox
sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in
C. glutamicum
reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/
loxP
system using a pair of mutant
lox
sites provides a new, efficient genome rearrangement technique for
C. glutamicum
. It should facilitate the understanding of genome functions of microorganisms.
Development of a Rapid Assay for Determining the Relative Abundance of Bacteria
Authors: Rowan Arlene K. , Davenport Russell J. , Snape Jason R. , Fearnside David , Barer Michael R. , Curtis Thomas P. , Head Ian M.
Published: December 2005

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 103to 104and 104to 105bacterial cells, respectively, forE. colirRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.
Horizontal Gene Transfer to Endogenous Endophytic Bacteria from Poplar Improves Phytoremediation of Toluene
Authors: Taghavi Safiyh , Barac Tanja , Greenberg Bill , Borremans Brigitte , Vangronsveld Jaco , van der Lelie Daniel
Published: December 2005

Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte
Burkholderia cepacia
VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium
B. cepacia
Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.
Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors
Authors: Mota Cesar , Head Melanie A. , Ridenoure Jennifer A. , Cheng Jay J. , de los Reyes Francis L.
Published: December 2005

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using
amoA
gene denaturant gradient gel electrophoresis.
Nitrosomonas
and
Nitrosococcus mobilis
were the dominant AOB and
Nitrospira
spp. were the dominant NOB in all reactors, although
Nitrosospira
and
Nitrobacter
were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest
Nitrosospira
rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of
Nitrobacter
, compared to the other reactors.
Nitrosomonas
sp. strain Nm107 was detected in all reactors, regardless of the reactor's performance. Close relatives of
Nitrosomonas europaea
,
Nitrosomonas
sp. strain ENI-11, and
Nitrosospira multiformis
were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.
Effect of Iron on Growth and Ultrastructure of
Acaryochloris marina
Authors: Swingley Wesley D. , Hohmann-Marriott Martin F. , Le Olson Tien , Blankenship Robert E.
Published: December 2005

The cyanobacterial genus
Acaryochloris
is the only known group of oxygenic phototrophs that contain chlorophyll
d
rather than chlorophyll
a
as the major photosynthetic pigment. Studies on this organism are still in their earliest stages, and biochemical analysis has rapidly outpaced growth optimization. We have investigated culture growth of the major strains of
Acaryochloris marina
(MBIC11017 and MBIC10697) by using several published and some newly developed growth media. It was determined that heavy addition of iron significantly enhanced culture longevity. These high-iron cultures showed an ultrastructure with thylakoid stacks that resemble traditional cyanobacteria (unlike previous studies). These cultures also show a novel reversal in the pigment ratios of the photosystem II signature components chlorophyll
a
and pheophytin
a
, as opposed to those in previous studies.
Interactions between the Fe(III)-Reducing Bacterium
Geobacter sulfurreducens
and Arsenate, and Capture of the Metalloid by Biogenic Fe(II)
Authors: Islam F. S. , Pederick R. L. , Gault A. G. , Adams L. K. , Polya D. A. , Charnock J. M. , Lloyd J. R.
Published: December 2005

Previous work has shown that microbial communities in As-mobilizing sediments from West Bengal were dominated by
Geobacter
species. Thus, the potential of
Geobacter sulfurreducens
to mobilize arsenic via direct enzymatic reduction and indirect mechanisms linked to Fe(III) reduction was analyzed.
G. sulfurreducens
was unable to conserve energy for growth via the dissimilatory reduction of As(V), although it was able to grow in medium containing fumarate as the terminal electron acceptor in the presence of 500 μM As(V). There was also no evidence of As(III) in culture supernatants, suggesting that resistance to 500 μM As(V) was not mediated by a classical arsenic resistance operon, which would rely on the intracellular reduction of As(V) and the efflux of As(III). When the cells were grown using soluble Fe(III) as an electron acceptor in the presence of As(V), the Fe(II)-bearing mineral vivianite was formed. This was accompanied by the removal of As, predominantly as As(V), from solution. Biogenic siderite (ferrous carbonate) was also able to remove As from solution. When the organism was grown using insoluble ferrihydrite as an electron acceptor, Fe(III) reduction resulted in the formation of magnetite, again accompanied by the nearly quantitative sorption of As(V). These results demonstrate that
G. sulfurreducens
, a model Fe(III)-reducing bacterium, did not reduce As(V) enzymatically, despite the apparent genetic potential to mediate this transformation. However, the reduction of Fe(III) led to the formation of Fe(II)-bearing phases that are able to capture arsenic species and could act as sinks for arsenic in sediments.
Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by
Dechloromonas
Strain RCB
Authors: Chakraborty Romy , O'Connor Susan M. , Chan Emily , Coates John D.
Published: December 2005

Dechloromonas
strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (
ortho
-,
meta
-, and
para
-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO
2
, strain RCB did not completely mineralize
para
-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 μM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.
Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on
Staphylococcus epidermidis
and
Staphylococcus haemolyticus
Biofilms
Authors: Cerca Nuno , Martins Silvia , Sillankorva Sanna , Jefferson Kimberly K. , Pier Gerald B. , Oliveira Rosario , Azeredo Joana
Published: December 2005

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative
Staphylococcus
species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-
N
-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in
Staphylococcus epidermidis
there were decreases in the oxygen and nitrogen contents, in
Staphylococcus haemolyticus
there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.