bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbon Isotope Fractionation during Anaerobic Degradation of Methyl
tert
-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions
Authors: Somsamak Piyapawn , Richnow Hans H. , Häggblom Max M.
Published: February 2006

Methyl
tert
-butyl ether (MTBE), an octane enhancer and a fuel oxygenate in reformulated gasoline, has received increasing public attention after it was detected as a major contaminant of water resources. Although several techniques have been developed to remediate MTBE-contaminated sites, the fate of MTBE is mainly dependent upon natural degradation processes. Compound-specific stable isotope analysis has been proposed as a tool to distinguish the loss of MTBE due to biodegradation from other physical processes. Although MTBE is highly recalcitrant, anaerobic degradation has been demonstrated under different anoxic conditions and may be an important process. To accurately assess in situ MTBE degradation through carbon isotope analysis, carbon isotope fractionation during MTBE degradation by different cultures under different electron-accepting conditions needs to be investigated. In this study, carbon isotope fractionation during MTBE degradation under sulfate-reducing and methanogenic conditions was studied in anaerobic cultures enriched from two different sediments. Significant enrichment of
13
C in residual MTBE during anaerobic biotransformation was observed under both sulfate-reducing and methanogenic conditions. The isotopic enrichment factors (ε) estimated for each enrichment were almost identical (−13.4

to −14.6

;
r
2
= 0.89 to 0.99). A ε value of −14.4

± 0.7

was obtained from regression analysis (
r
2
= 0.97,
n
= 55, 95% confidence interval), when all data from our MTBE-transforming anaerobic cultures were combined. The similar magnitude of carbon isotope fractionation in all enrichments regardless of culture or electron-accepting condition suggests that the terminal electron-accepting process may not significantly affect carbon isotope fractionation during anaerobic MTBE degradation.
Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures ofEscherichia coli
Authors: Nanchen Annik , Schicker Alexander , Sauer Uwe
Published: February 2006

A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic13C analysis of intracellular fluxes over a broad range of growth rates in glucose-limitedEscherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h−1. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO2. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h−1was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.
Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms
Authors: Mazzeo Maria Fiorella , Sorrentino Alida , Gaita Marcello , Cacace Giuseppina , Di Stasio Michele , Facchiano Angelo , Comi Giuseppe , Malorni Antonio , Siciliano Rosa Anna
Published: February 2006

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify
Escherichia coli
O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.
Coexistence of
Burkholderia
,
Cupriavidus
, and
Rhizobium
sp. Nodule Bacteria on two
Mimosa
spp. in Costa Rica
Authors: Barrett Craig F. , Parker Matthew A.
Published: February 2006

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two
Mimosa
species at three sites in Costa Rica belonged to the genera
Burkholderia
,
Cupriavidus
, and
Rhizobium
. This is the first report of
Cupriavidus
sp. nodule symbionts for
Mimosa
populations within their native geographic range in the neotropics.
Burkholderia
spp. predominated among samples from
Mimosa pigra
(86% of isolates), while there was a more even distribution of
Cupriavidus
,
Burkholderia
, and
Rhizobium
spp. on
Mimosa pudica
(38, 37, and 25% of isolates, respectively). All
Cupriavidus
and
Burkholderia
genotypes tested formed root nodules and fixed nitrogen on both
M. pigra
and
M. pudica
, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both
Cupriavidus
and
Burkholderia
spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of
Rhizobium
. Given the prevalence of
Burkholderia
and
Cupriavidus
spp. on these
Mimosa
legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both β-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.
Characterization of the
tre
Locus and Analysis of Trehalose Cryoprotection in
Lactobacillus acidophilus
NCFM
Authors: Duong Tri , Barrangou Rodolphe , Russell W. Michael , Klaenhammer Todd R.
Published: February 2006

Freezing and lyophilization are common methods used for preservation and storage of microorganisms during the production of concentrated starter cultures destined for industrial fermentations or product formulations. The compatible solute trehalose has been widely reported to protect bacterial, yeast and animal cells against a variety of environmental stresses, particularly freezing and dehydration. Analysis of the
Lactobacillus acidophilus
NCFM genome revealed a putative trehalose utilization locus consisting of a transcriptional regulator,
treR
; a trehalose phosphoenolpyruvate transferase system (PTS) transporter,
treB
; and a trehalose-6-phosphate hydrolase,
treC
. The objective of this study was to characterize the
tre
locus in
L. acidophilus
and determine whether or not intracellular uptake of trehalose contributes to cryoprotection. Cells subjected to repeated freezing and thawing cycles were monitored for survival in the presence of various concentrations of trehalose. At 20% trehalose a 2-log increase in survival was observed. The trehalose PTS transporter and trehalose hydrolase were disrupted by targeted plasmid insertions. The resulting mutants were unable to grow on trehalose, indicating that both trehalose transport into the cell via a PTS and hydrolysis via a trehalose-6-phosphate hydrolase were necessary for trehalose fermentation. Trehalose uptake was found to be significantly reduced in the transporter mutant but unaffected in the hydrolase mutant. Additionally, the cryoprotective effect of trehalose was reduced in these mutants, suggesting that intracellular transport and hydrolysis contribute significantly to cryoprotection.
Sequence Variation among Group III F-Specific RNA Coliphages from Water Samples and Swine Lagoons
Authors: Stewart Jill R. , Vinjé Jan , Oudejans Sjon J. G. , Scott Geoff I. , Sobsey Mark D.
Published: February 2006

Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5′ end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Q-beta. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Q-beta-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Q-beta-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications.
Isolation and Characterization of a Cyanophage Infecting the Toxic Cyanobacterium
Microcystis aeruginosa
Authors: Yoshida Takashi , Takashima Yukari , Tomaru Yuji , Shirai Yoko , Takao Yoshitake , Hiroishi Shingo , Nagasaki Keizo
Published: February 2006

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium
Microcystis aeruginosa
. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of
Microcystis
blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.
Catabolism of 1,5-Anhydro-d-Fructose inSinorhizobium morelenseS-30.7.5: Discovery, Characterization, and Overexpression of a New 1,5-Anhydro-d-Fructose Reductase and Its Application in Sugar Analysis and Rare Sugar Synthesis
Authors: Kühn Annette , Yu Shukun , Giffhorn Friedrich
Published: February 2006

The bacteriumSinorhizobium morelenseS-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-d-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-d-mannitol (AM) and the further conversion of AM tod-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined toS. morelenseS-30.7.5 but is a more common feature among theRhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frameSmc04400localized on the chromosome ofSinorhizobium meliloti1021. Based on sequence information for this locus, theafrgene was cloned fromS. morelenseS-30.7.5 and overexpressed inEscherichia coli. In addition to the oxidoreductase ofS. meliloti1021, AFR showed high sequence similarities to putative oxidoreductases ofMesorhizobium loti,Brucella suis, andB. melitensisbut not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.
Root-Associated Bacteria Contribute to Mineral Weathering and to Mineral Nutrition in Trees: a Budgeting Analysis
Authors: Calvaruso Christophe , Turpault Marie-Pierre , Frey-Klett Pascale
Published: February 2006

The principal nutrient source for forest trees derives from the weathering of soil minerals which results from water circulation and from plant and microbial activity. The main objectives of this work were to quantify the respective effects of plant- and root-associated bacteria on mineral weathering and their consequences on tree seedling growth and nutrition. That is why we carried out two column experiments with a quartz-biotite substrate. The columns were planted with or without pine seedlings and inoculated or not with three ectomycorrhizosphere bacterial strains to quantify biotite weathering and pine growth and to determine how bacteria improve pine growth. We showed that the pine roots significantly increased biotite weathering by a factor of 1.3 for magnesium and 1.7 for potassium. We also demonstrated that the inoculation of
Burkholderia glathei
PML1(12) significantly increased biotite weathering by a factor of 1.4 for magnesium and 1.5 for potassium in comparison with the pine alone. In addition, we observed a significant positive effect of
B. glathei
PMB1(7) and PML1(12) on pine growth and on root morphology (number of lateral roots and root hairs). We demonstrated that PML1(12) improved pine growth when the seedlings were supplied with a nutrient solution which did not contain the nutrients present in the biotite. No improvement of pine growth was observed when the seedlings were supplied with all the nutrients necessary for pine growth. We therefore propose that the growth-promoting effect of
B. glathei
PML1(12) mainly resulted from the improved plant nutrition via increased mineral weathering.
RNA-Mediated Gene Silencing in Monokaryons and Dikaryons of
Schizophyllum commune
Authors: de Jong Jan F. , Deelstra Heine J. , Wösten Han A. B. , Lugones Luis G.
Published: February 2006

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete
Schizophyllum commune
. For instance, the
SC3
and
SC15
genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The
SC15
gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of
S. commune
after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.
Construction and Complementation of In-Frame Deletions of the Essential
Escherichia coli
Thymidylate Kinase Gene
Authors: Chaperon David-Nicolas
Published: February 2006

This work reports the construction of
Escherichia coli
in-frame deletion strains of
tmk
, which encodes thymidylate kinase, Tmk. The
tmk
gene is located at the third position of a putative five-gene operon at 24.9 min on the
E. coli
chromosome, which comprises the genes
pabC
,
yceG
,
tmk
,
holB
, and
ycfH
. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded
tmk
, the
tmk
gene was replaced by the kanamycin resistance gene
kka1
, encoding amino glycoside 3′-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The
E. coli tmk
gene is essential under any conditions tested. To show functional complementation in bacteria, the
E. coli tmk
gene was replaced by thymidylate kinases of bacteriophage T4 gp
1
,
E. coli tmk
,
Saccharomyces cerevisiae cdc8
, or the
Homo sapiens
homologue,
dTYMK
. Growth of these transgenic
E. coli
strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes
holB
and
ycfH
with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.
Cloning and Functional Characterization of the
styE
Gene, Involved in Styrene Transport in
Pseudomonas putida
CA-3
Authors: Mooney Aisling , O'Leary Niall D. , Dobson Alan D. W.
Published: February 2006

A 1.5-kb region immediately downstream of the
styABCD
operon involved in styrene degradation in
Pseudomonas putida
CA-3 has been cloned. Sequence analysis revealed a 1,296-bp open reading frame, designated
styE
, and BLAST P database comparisons of the deduced StyE amino acid sequence revealed 33 to 98% identity with several membrane-associated ATPase-dependent kinase proteins involved in the active transport of aromatic hydrocarbons across bacterial membranes and also with FadL, an outer membrane protein necessary for the uptake of long-chain fatty acids in
Escherichia coli
. Transcription of
styE
is styrene dependent, and the gene is cotranscribed with the
styABCD
structural genes. StyE appears to be membrane associated, with a corresponding 45.9-kDa band being identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane preparations from styrene-grown cells.
P. putida
CA-3 cells in which the
styE
gene had been interrupted were no longer capable of growth on styrene. In contrast, overexpression of
styE
in
P. putida
CA-3 resulted in a 4.2-fold increase in styrene monooxygenase activity compared with wild-type cells grown on styrene, with a concomitant 8-fold increase in
styA
mRNA transcript levels. Experiments with the classic, ATPase inhibitor vanadate revealed that growth of wild-type cells on styrene was inhibited at a concentration of 1 mM, while 1.75 mM was required to achieve a similar effect in the StyE overexpression strain. Growth of either strain on citrate was not inhibited in the presence of up to 7 mM vanadate. These findings suggest a role for StyE in the active transport of styrene in
Pseudomonas putida
CA-3 and identify styrene transport as a potentially limiting factor with respect to mRNA transcript levels and associated enzymatic activity of the styrene degradative pathway.
Genetic Modification of
Bacillus subtilis
for Production of
d
-
chiro
-Inositol, an Investigational Drug Candidate for Treatment of Type 2 Diabetes and Polycystic Ovary Syndrome
Authors: Yoshida Ken-ichi , Yamaguchi Masanori , Morinaga Tetsuro , Ikeuchi Maya , Kinehara Masaki , Ashida Hitoshi
Published: February 2006

d
-
chiro
-Inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of
Bacillus subtilis
for production of DCI from
myo
-inositol (MI). The
B. subtilis iolABCDEFGHIJ
operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3
d
-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified
iolI
encoding inosose isomerase, which converts 2KMI to 1-keto-
d
-
chiro
-inositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of
iolE
in a mutant constitutively expressing the
iol
operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.
Natural Transformation-Mediated Transfer of Erythromycin Resistance in
Campylobacter coli
Strains from Turkeys and Swine
Authors: Kim Joo-Sung , Carver Donna K. , Kathariou Sophia
Published: February 2006

Erythromycin resistance in
Campylobacter coli
from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in
C. coli
remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse
C. coli
strains from turkeys and swine, with total genomic DNA from erythromycin-resistant
C. coli
of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in
C. coli
strains from turkeys than in swine-derived strains (
P
< 0.01). The frequency of transformation to erythromycin resistance was 10
−5
to 10
−6
for turkey-derived strains but 10
−7
or less for
C. coli
from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the
C. coli
donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among
C. coli
strains colonizing meat animals.
Evidence for Autotrophy via the Reverse Tricarboxylic Acid Cycle in the Marine Magnetotactic Coccus Strain MC-1
Authors: Williams Timothy J. , Zhang Chuanlun L. , Scott James H. , Bazylinski Dennis A.
Published: February 2006

Strain MC-1 is a marine, microaerophilic, magnetite-producing, magnetotactic coccus phylogenetically affiliated with the α-
Proteobacteria
. Strain MC-1 grew chemolithotrophically with sulfide and thiosulfate as electron donors with HCO
3
−
/CO
2
as the sole carbon source. Experiments with cells grown microaerobically in liquid with thiosulfate and H
14
CO
3
−
/
14
CO
2
showed that all cell carbon was derived from H
14
CO
3
−
/
14
CO
2
and therefore that MC-1 is capable of chemolithoautotrophy. Cell extracts did not exhibit ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) activity, nor were RubisCO genes found in the draft genome of MC-1. Thus, unlike other chemolithoautotrophic, magnetotactic bacteria, strain MC-1 does not appear to utilize the Calvin-Benson-Bassham cycle for autotrophy. Cell extracts did not exhibit carbon monoxide dehydrogenase activity, indicating that the acetyl-coenzyme A pathway also does not function in strain MC-1. The
13
C content of whole cells of MC-1 relative to the
13
C content of the inorganic carbon source (Δδ
13
C) was −11.4

. Cellular fatty acids showed enrichment of
13
C relative to whole cells. Strain MC-1 cell extracts showed activities for several key enzymes of the reverse (reductive) tricarboxylic acid (rTCA) cycle including fumarate reductase, pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor oxidoreductase. Although ATP citrate lyase (another key enzyme of the rTCA cycle) activity was not detected in strain MC-1 using commonly used assays, cell extracts did cleave citrate, and the reaction was dependent upon the presence of ATP and coenzyme A. Thus, we infer the presence of an ATP-dependent citrate-cleaving mechanism. These results are consistent with the operation of the rTCA cycle in MC-1. Strain MC-1 appears to be the first known representative of the α-
Proteobacteria
to use the rTCA cycle for autotrophy.
Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C
Authors: Dumont Frédéric , Marechal Pierre-André , Gervais Patrick
Published: February 2006

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.
Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded byThermotoga maritimaBind Sugars
Authors: Nanavati Dhaval M. , Thirangoon Kamolwan , Noll Kenneth M.
Published: February 2006

The hyperthermophilic bacteriumThermotoga maritimahas shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the β-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), β-1,4-mannobiose; and TM1226 (ManD), β-1,4-mannobiose, β-1,4-mannotriose, β-1,4-mannotetraose, β-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE),myo-inositol; TM0432 (AguE), α-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found thatT. maritimadoes not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.
Unstable Lysogeny and Pseudolysogeny in
Vibrio harveyi
Siphovirus-Like Phage 1
Authors: Khemayan Krit , Pasharawipas Tirasak , Puiprom Orapim , Sriurairatana Siriporn , Suthienkul Orasa , Flegel Timothy W.
Published: February 2006

Exposure of
Vibrio harveyi
(strain VH1114) to
V. harveyi
siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.
Identification of a
Thiomicrospira denitrificans
-Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea
Authors: Brettar Ingrid , Labrenz Matthias , Flavier Sébastien , Bötel Julia , Kuosa Harri , Christen Richard , Höfle Manfred G.
Published: February 2006

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier
Thiomicrospira denitrificans
. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of
T. denitrificans
. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to
T. denitrificans
could be relevant for the carbon budget of the central Baltic Sea because
T. denitrificans
exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of
Thiobacillus denitrificans
hypothesized previously for this function.
Neutral Lipid Biosynthesis in Engineered
Escherichia coli
: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters
Authors: Kalscheuer Rainer , Stöveken Tim , Luftmann Heinrich , Malkus Ursula , Reichelt Rudolf , Steinbüchel Alexander
Published: February 2006

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (
Simmondsia chinensis
) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant
Escherichia coli
strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from
Acinetobacter baylyi
strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant
E. coli
were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in
E. coli
, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.
Comparison of the Efficacies of Disinfectants To Control Microbial Contamination in Dental Unit Water Systems in General Dental Practices across the European Union
Authors: Schel A. J. , Marsh P. D. , Bradshaw D. J. , Finney M. , Fulford M. R. , Frandsen E. , Østergaard E. , ten Cate J. M. , Moorer W. R. , Mavridou A. , Kamma J. J. , Mandilara G. , Stösser L. , Kneist S. , Araujo R. , Contreras N. , Goroncy-Bermes P. , O'Mullane D. , Burke F. , O'Reilly P. , Hourigan G. , O'Sullivan M. , Holman R. , Walker J. T.
Published: February 2006

Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Association's guideline of <200 CFU · ml−1for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n= 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU · ml−1. Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU · ml−1, although occasional high values (up to 4.88 log CFU · ml−1) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.
Glutamate Dehydrogenase Activity Can Be Transmitted Naturally to
Lactococcus lactis
Strains To Stimulate Amino Acid Conversion to Aroma Compounds
Authors: Tanous Catherine , Chambellon Emilie , Le Bars Dominique , Delespaul Gilbert , Yvon Mireille
Published: February 2006

Amino acid conversion to aroma compounds by
Lactococcus lactis
is limited by the low production of α-ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to α-ketoglutarate was detected in
L. lactis
strains isolated from a vegetal source, and the gene responsible for the activity in
L. lactis
NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study,
gdh
gene inactivation and overexpression confirmed the direct impact of GDH activity of
L. lactis
on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying
gdh
was naturally transmitted to another
L. lactis
strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.
Degradation of 2,3-Diethyl-5-Methylpyrazine by a Newly Discovered Bacterium,
Mycobacterium
sp. Strain DM-11
Authors: Rappert Sugima , Botsch Kathrin Caroline , Nagorny Stephanie , Francke Wittko , Müller Rudolf
Published: February 2006

A bacterium was isolated from the waste gas treatment plant at a fishmeal processing company on the basis of its capacity to use 2,3-diethyl-5-methylpyrazine (DM) as a sole carbon and energy source. The strain, designated strain DM-11, grew optimally at 25°C and had a doubling time of 29.2 h. The strain did not grow on complex media like tryptic soy broth, Luria-Bertani broth, or nutrient broth or on simple carbon sources like glucose, acetate, oxoglutarate, succinate, or citrate. Only on Löwenstein-Jensen medium was growth observed. The 16S rRNA gene sequence of strain DM-11 showed the highest similarity (96.2%) to
Mycobacterium poriferae
strain ATCC 35087
T
. Therefore, strain DM-11 merits recognition as a novel species within the genus
Mycobacterium.
DM also served as a sole nitrogen source for the growth of strain DM-11. The degradation of DM by strain DM-11 requires molecular oxygen. The first intermediate was identified as 5,6-diethyl-2-hydroxy-3-methylpyrazine (DHM). Its disappearance was accompanied by the release of ammonium into the culture medium. No other metabolite was detected. We conclude that ring fission occurred directly after the formation of DHM and ammonium was eliminated after ring cleavage. Molecular oxygen was essential for the degradation of DHM. The expression of enzymes involved in the degradation of DM and DHM was regulated. Only cells induced by DM or DHM converted these compounds. Strain DM-11 also grew on 2-ethyl-5(6)-methylpyrazine (EMP) and 2,3,5-trimethylpyrazine (TMP) as a sole carbon, nitrogen, and energy source. In addition, the strain converted many pyrazines found in the waste gases of food industries cometabolically.
Phosphorus Scavenging in the Unicellular Marine Diazotroph
Crocosphaera watsonii
Authors: Dyhrman Sonya T. , Haley Sheean T.
Published: February 2006

Through the fixation of atmospheric nitrogen and photosynthesis, marine diazotrophs play a critical role in the global cycling of nitrogen and carbon.
Crocosphaera watsonii
is a recently described unicellular diazotroph that may significantly contribute to marine nitrogen fixation in tropical environments. One of the many factors that can constrain the growth and nitrogen fixation rates of marine diazotrophs is phosphorus bioavailability. Using genomic and physiological approaches, we examined phosphorus scavenging mechanisms in strains of
C. watsonii
from both the Atlantic and the Pacific. Observations from the
C. watsonii
WH8501 genome suggest that this organism has the capacity for high-affinity phosphate transport (e.g., homologs of
pstSCAB
) in low-phosphate, oligotrophic systems. The
pstS
gene (high-affinity phosphate binding) is present in strains isolated from both the Atlantic and the Pacific, and its expression was regulated by the exogenous phosphate supply in strain WH8501. Genomic observation also indicated a broad capacity for phosphomonoester hydrolysis (e.g., a putative alkaline phosphatase). In contrast, no clear homologs of genes for phosphonate transport and hydrolysis could be identified. Consistent with these genomic observations,
C. watsonii
WH8501 is able to grow on phosphomonoesters as a sole source of added phosphorus but not on the phosphonates tested to date. Taken together these data suggest that
C. watsonii
has a robust capacity for scavenging phosphorus in oligotrophic systems, although this capacity differs from that of other marine cyanobacterial genera, such as
Synechococcus
,
Prochlorococcus
, and
Trichodesmium
.
Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva
Authors: Wescombe Philip A. , Upton Mathew , Dierksen Karen P. , Ragland Nancy L. , Sivabalan Senthuran , Wirawan Ruth E. , Inglis Megan A. , Moore Chris J. , Walker Glenn V. , Chilcott Chris N. , Jenkinson Howard F. , Tagg John R.
Published: February 2006

Salivaricin A (SalA), the first
Streptococcus salivarius
lantibiotic to be characterized, appears to be inhibitory to most
Streptococcus pyogenes
strains. A variant of the SalA structural gene (
salA1
) is present in more than 90% of
S. pyogenes
strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (
salA2
to
salA5
) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against
Micrococcus luteus
and in their ability to act as inducers of SalA production. It had previously been shown that
S. pyogenes
strain SF370 had a deletion (of approximately 2.5 kb) in the
salM
and
salT
genes of the
salA1
locus. In the present study, several additional characteristic deletions within the
salA1
loci were identified.
S. pyogenes
strains of the same M serotype all share the same
salA1
locus structure. Since
S. salivarius
is a predominant member of the normal oral flora of healthy humans, strains producing anti-
S. pyogenes
lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing
S. salivarius
or who have been colonized with the SalA2-producing probiotic
S. salivarius
K12.
A Novel
Bacteroidetes
Symbiont Is Localized in
Scaphoideus titanus
, the Insect Vector of Flavescence Dorée in
Vitis vinifera
Authors: Marzorati Massimo , Alma Alberto , Sacchi Luciano , Pajoro Massimo , Palermo Simona , Brusetti Lorenzo , Raddadi Noura , Balloi Annalisa , Tedeschi Rosemarie , Clementi Emanuela , Corona Silvia , Quaglino Fabio , Bianco Piero Attilio , Beninati Tiziana , Bandi Claudio , Daffonchio Daniele
Published: February 2006

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “
Candidatus
Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper,
Scaphoideus titanus
(Cicadellidae). After collection of
S. titanus
specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with
Bacteroidetes
symbionts of the “
Candidatus
Cardinium hertigii” group. In addition, electron microscopy of tissues of
S. titanus
fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “
Ca.
Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “
Ca.
Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “
Ca.
Phytoplasma vitis” have a complex life cycle in the body of
S. titanus
and are colocalized in different organs and tissues.
Degradation of 4-Chloro-2-Methylphenoxyacetic Acid in Top- and Subsoil Is Quantitatively Linked to the Class III
tfdA
Gene
Authors: Bælum Jacob , Henriksen Trine , Hansen Hans Christian Bruun , Jacobsen Carsten Suhr
Published: February 2006

The
tfdA
gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other
tfdA
-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of
tfdA
genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the
tfdA
-containing bacterial community, from 500 genes g
−1
soil to approximately 3 × 10
4
genes g
−1
soil and to 7 × 10
5
genes g
−1
soil for topsoil initially added to 2.3 mg MCPA kg
−1
(dry weight) soil and 20 mg MCPA kg
−1
(dry weight) soil, respectively. We analyzed the diversity of the
tfdA
gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant
tfdA
population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the
tfdA
genes responsible for the degradation of MCPA belonged to the class III
tfdA
genes, while the
tfdA
genes present in the soil before the occurrence of degradation belonged to the class I
tfdA
genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.