bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in
Saccharomyces cerevisiae
Authors: Sonderegger Marco , Schümperli Michael , Sauer Uwe
Published: May 2004

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with
Saccharomyces cerevisiae
. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting
Saccharomyces cerevisiae
strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the
ald6
mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.
Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered
Saccharomyces cerevisiae
: Possible Consequence of Energy-Dependent Lactate Export
Authors: van Maris Antonius J. A. , Winkler Aaron A. , Porro Danilo , van Dijken Johannes P. , Pronk Jack T.
Published: May 2004

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents.
Saccharomyces cerevisiae
(CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative
S. cerevisiae
strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.
Structural and Functional Differences in Two Cyclic Bacteriocins with the Same Sequences Produced by Lactobacilli
Authors: Kawai Yasushi , Ishii Yasuyuki , Arakawa Kensuke , Uemura Koichiro , Saitoh Boku , Nishimura Junko , Kitazawa Haruki , Yamazaki Yukiko , Tateno Yoshio , Itoh Takatoshi , Saito Tadao
Published: May 2004

Lactobacillus gasseri
LA39 and
L. reuteri
LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of
L. reuteri
LA6, but reutericin 6 did not inhibit the growth of
L. gasseri
LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of α-helices, the spectra of the bacteriocins did not coincide. The results of
d
- and
l
-amino acid composition analysis showed that two residues and one residue of
d
-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different
d
-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in
d
-amino acid contents in the molecules.
Molecular and Physiological Analysis of the Role of Osmolyte Transporters BetL, Gbu, and OpuC in Growth of
Listeria monocytogenes
at Low Temperatures
Authors: Wemekamp-Kamphuis Henrike H. , Sleator Roy D. , Wouters Jeroen A. , Hill Colin , Abee Tjakko
Published: May 2004

Listeria monocytogenes
is a ubiquitous food-borne pathogen found widely distributed in nature as well as an undesirable contaminant in a variety of fresh and processed foods. This ubiquity can be at least partly explained by the ability of the organism to grow at high osmolarity and reduced temperatures, a consequence of its ability to accumulate osmo- and cryoprotective compounds termed osmolytes. Single and multiple deletions of the known osmolyte transporters BetL, Gbu, and OpuC significantly reduce growth at low temperatures. During growth in brain heart infusion broth at 7°C, Gbu and OpuC had a more pronounced role in cryoprotection than did BetL. However, upon the addition of betaine to defined medium, the hierarchy of transporter importance shifted to Gbu > BetL > OpuC. Upon the addition of carnitine, only OpuC appeared to play a role in cryoprotection. Measurements of the accumulated osmolytes showed that betaine is preferred over carnitine, while in the absence of a functional Gbu, carnitine was accumulated to higher levels than betaine was at 7°C. Transcriptional analysis of the genes encoding BetL, Gbu, and OpuC revealed that each transporter is induced to different degrees upon cold shock of
L. monocytogenes
LO28. Additionally, despite being transcriptionally up-regulated upon cold shock, a putative fourth osmolyte transporter, OpuB (identified by bioinformatic analysis and encoded by
lmo1421
and
lmo1422
), showed no significant contribution to listerial chill tolerance. Growth of the quadruple mutant LO28ΔBCGB (Δ
betL
Δ
opuC
Δ
gbu
Δ
opuB
) was comparable to the that of the triple mutant LO28ΔBCGsoe (Δ
betL
Δ
opuC
Δ
gbu
) at low temperatures. Here, we conclude that betaine and carnitine transport upon low-temperature exposure is mediated via three osmolyte transporters, BetL, Gbu, and OpuC.
Relative Neurotoxin Gene Expression inClostridium botulinumType B, Determined Using Quantitative Reverse Transcription-PCR
Authors: Lövenklev Maria , Holst Elisabet , Borch Elisabeth , Rådström Peter
Published: May 2004

A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) inClostridium botulinum. The levels ofcntBmRNA in five type B strains were accurately monitored by using primers specific forcntBand for the reference gene encoding the 16S rRNA. The patterns and relative expression ofcntBwere different in the different strains. Except for one of the strains investigated, an increase incntBexpression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strainC. botulinumATCC 7949, the level ofcntBmRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air oncntBexpression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level ofcntBmRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression ofcntBcompared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in thecntBmRNA level was observed in all growth phases.
Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic
Clostridium botulinum
Type B
Authors: Lövenklev Maria , Artin Ingrid , Hagberg Oskar , Borch Elisabeth , Holst Elisabet , Rådström Peter
Published: May 2004

The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (
cntB
) expression in nonproteolytic
Clostridium botulinum
were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative
cntB
expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO
2
in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the
cntB
-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO
2
was found to have a major effect on gene expression when the
cntB
mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of
cntB
relative to the expression of the 16S rRNA gene was stimulated by an elevated CO
2
concentration; the
cntB
mRNA level was fivefold greater in a 70% CO
2
atmosphere than in a 10% CO
2
atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml
−1
 · unit of optical density
−1
in the 10% CO
2
atmosphere and 126 ng · ml
−1
 · unit of optical density
−1
in the 70% CO
2
atmosphere.
Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds
Authors: Morasch Barbara , Richnow Hans H. , Vieth Andrea , Schink Bernhard , Meckenstock Rainer U.
Published: May 2004

Stable isotope fractionation was studied during the degradation of
m
-xylene,
o
-xylene,
m
-cresol, and
p
-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors (ε) of −1.5 and −3.9

, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic ε (ε
intrinsic
) were calculated. A comparison of ε
intrinsic
at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific ε elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of ε found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average ε if no fractionation factor is available for single compounds.
Viral Activity in Two Contrasting Lake Ecosystems
Authors: Bettarel Yvan , Sime-Ngando Télesphore , Amblard Christian , Dolan John
Published: May 2004

For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses. In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat. Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory. Overall, viral infection parameters were much less variable than bacterial production. We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October). FVIC values were significantly higher in Lake Pavin (mean [M] = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium
−1
, respectively. We detected no significant depth-related differences in FVIC or burst size. We found that in both lakes the removal of bacterial production by flagellate grazing (M
Pavin
= 37.7%, M
Aydat
= 18.5%) was nearly always more than the production removed by viral lysis (M
Pavin
= 16.2%, M
Aydat
= 19%) or ciliate grazing (M
Pavin
= 2.7%, M
Aydat
= 8.8%). However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat. In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers. Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system.
Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant,
Arthrobacter chlorophenolicus
A6
Authors: Backman Agneta , Maraha Ninwe , Jansson Janet K.
Published: May 2004

Arthrobacter chlorophenolicus
A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the
gfp
gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [
luc
]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make
A. chlorophenolicus
A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.
Isolation, Characterization, and U(VI)-Reducing Potential of a Facultatively Anaerobic, Acid-Resistant Bacterium from Low-pH, Nitrate- and U(VI)-Contaminated Subsurface Sediment and Description of
Salmonella subterranea
sp. nov
Authors: Shelobolina Evgenya S. , Sullivan Sara A. , O'Neill Kathleen R. , Nevin Kelly P. , Lovley Derek R.
Published: May 2004

A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 μm long and 0.7 to 0.9 μm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H
2
S production, and for gelatin hydrolysis. Strain FRCl was capable of using O
2
, NO
3
−
, S
2
O
3
2−
, fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4% similar to
Salmonella bongori
and 96.3% similar to
Enterobacter cloacae
. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus
Salmonella
and represents a new species,
Salmonella subterranea
sp. nov.
Metal Toxicity Affects Fungal and Bacterial Activities in Soil Differently
Authors: Rajapaksha R. M. C. P. , Tobor-Kapłon M. A , Bååth E.
Published: May 2004

Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load.
Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of
Acinetobacter
sp. Strain ADP1
Authors: Parke Donna , Ornston L. Nicholas
Published: May 2004

Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the
hca
genes in the nutritionally versatile, naturally transformable bacterium
Acinetobacter
sp. strain ADP1. A key step in the
hca
-encoded pathway is activation of the natural substrates caffeate,
p
-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by
hcaC
. As described in this paper,
Acinetobacter
cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates;
Escherichia coli
cells carrying a subclone with the
hcaC
gene are hydroxycinnamate sensitive as well. When the
hcaA
mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated
Acinetobacter
cells to caffeate,
p
-coumarate, or ferulate at 10
−6
M totally inhibited the growth of cells. The toxicity of
p
-coumarate and ferulate to a Δ
hcaA
strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an
Acinetobacter
strain blocked in
hcaA
, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of
hcaC
led to the development of a positive-selection strategy that targets mutations blocking HcaC. An
hcaC
mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.
Novel Polyketide Synthase from
Nectria haematococca
Authors: Graziani Stephane , Vasnier Christelle , Daboussi Marie-Josee
Published: May 2004

We identified a polyketide synthase (PKS) gene,
pksN
, from a strain of
Nectria haematococca
by complementing a mutant unable to synthesize a red perithecial pigment.
pksN
encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: β-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The
pksN
product groups with the
Aspergillus nidulans
WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of
pksN
did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that
pksN
encodes a novel PKS required for the perithecial red pigment biosynthesis.
Survival and Dormancy ofMycobacterium aviumsubsp.paratuberculosisin theEnvironment
Authors: Whittington Richard J. , Marshall D. Jeff , Nicholls Paul J. , Marsh Ian B. , Reddacliff Leslie A.
Published: May 2004

The survival ofMycobacterium aviumsubsp.paratuberculosiswas studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. Adps-like genetic element andrelA, which are involved in dormancy responses in other mycobacteria, are present in theM. aviumsubsp.paratuberculosisgenome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival ofM. aviumsubsp.paratuberculosisin the environment is finite, consistent with its taxonomic description as an obligate parasite of animals.
Studies of Single-Chain Antibody Expression in Quiescent
Escherichia coli
Authors: Mukherjee K. J. , Rowe D. C. D. , Watkins N. A. , Summers D. K.
Published: May 2004

Quiescent
Escherichia coli
cells are generated by overexpressing the Rcd transcript in an
hns
-
205
mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λP
L
under the control of the
c
I857 temperature-sensitive repressor, while the second expresses Rcd from λP
R
. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD
600
] of 3.5) was 37 mg liter
−1
, compared to a maximum of 13 mg liter
−1
in the control culture (final OD
600
of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD
600
of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter
−1
. A control culture with a similar feed reached an OD
600
of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter
−1
. Quiescent cells at an OD
600
of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter
−1
was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent
E. coli
cultures that are capable of efficient synthesis, folding, and export of proteins.
Genomic Diversity of
Erwinia carotovora
subsp.
carotovora
and Its Correlation with Virulence
Authors: Mee-Ngan Yap , Barak Jeri D. , Charkowski Amy O.
Published: May 2004

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen
Erwinia carotovora
subsp.
carotovora
. The results obtained with each method showed that
E. carotovora
subsp.
carotovora
strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial
mdh
sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for
Escherichia coli.
Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven
rrn
operons in all
E. carotovora
subsp.
carotovora
strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant polymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain
E. carotovora
subsp.
carotovora
71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPP17, failed to oxidize cellobiose and was missing several
hrp
and
hrc
genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen.
Particle-Based Multidimensional Multispecies Biofilm Model
Authors: Picioreanu Cristian , Kreft Jan-Ulrich , van Loosdrecht Mark C. M.
Published: May 2004

In this paper we describe a spatially multidimensional (two-dimensional [2-D] and three-dimensional [3-D]) particle-based approach for modeling the dynamics of multispecies biofilms growing on multiple substrates. The model is based on diffusion-reaction mass balances for chemical species coupled with microbial growth and spreading of biomass represented by hard spherical particles. Effectively, this is a scaled-up version of a previously proposed individual-based biofilm model. Predictions of this new particle-based model were quantitatively compared with those obtained with an established one-dimensional (1-D) multispecies model for equivalent problems. A nitrifying biofilm containing aerobic ammonium and nitrite oxidizers, anaerobic ammonium oxidizers, and inert biomass was chosen as an example. The 2-D and 3-D models generally gave the same results. If only the average flux of nutrients needs to be known, 2-D and 1-D models are very similar. However, the behavior of intermediates, which are produced and consumed in different locations within the biofilm, is better described in 2-D and 3-D models because of the multidirectional concentration gradients. The predictions of 2-D or 3-D models are also different from those of 1-D models for slowly growing or minority species in the biofilm. This aspect is related to the mechanism of biomass spreading or advection implemented in the models and should receive more attention in future experimental studies.
Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement
Authors: Garrett James B. , Kretz Keith A. , O'Donoghue Eileen , Kerovuo Janne , Kim William , Barton Nelson R. , Hazlewood Geoffrey P. , Short Jay M. , Robertson Dan E. , Gray Kevin A.
Published: May 2004

The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (
myo
-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the
appA
gene from
Escherichia coli
, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the
appA
parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.
Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection
Authors: Vora Gary J. , Meador Carolyn E. , Stenger David A. , Andreadis Joanne D.
Published: May 2004

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, φ29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic
Escherichia coli
O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and φ29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.
Comparative Analysis of the Conventional and Novel
pmo
(Particulate Methane Monooxygenase) Operons from
Methylocystis
Strain SC2
Authors: Ricke Peter , Erkel Christoph , Kube Michael , Reinhardt Richard , Liesack Werner
Published: May 2004

In addition to the conventional
pmoA
gene (
pmoA1
) encoding the active site polypeptide of particulate methane monooxygenase, a novel
pmoA
gene copy (
pmoA2
) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the
pmoA1
and
pmoA2
gene copies in the type II MOB
Methylocystis
strain SC2 are each part of a complete
pmoCAB
gene cluster (
pmoCAB1
,
pmoCAB2
). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either
pmoCAB1
or
pmoCAB2
were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The
amoCAB
gene cluster encodes ammonia monooxygenase and is evolutionarily related to
pmoCAB
. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that
pmoCAB2
encodes a functional equivalent of PmoCAB1. In experiments in which we used 5′ rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of
pmoC1
and
pmoC2
, respectively. Immediately upstream of the transcriptional start sites of both
pmoCAB1
and
pmoCAB2
, sequence motifs similar to
Escherichia coli
σ
70
promoters were identified.
Growth of Polychlorinated-Biphenyl-Degrading Bacteria in the Presence of Biphenyl and Chlorobiphenyls Generates Oxidative Stress and Massive Accumulation of Inorganic Polyphosphate
Authors: Chávez Francisco P. , Lünsdorf Heinrich , Jerez Carlos A.
Published: May 2004

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium
Pseudomonas
sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.
In Vivo Study of
Trichoderma
-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems
Authors: Lu Zexun , Tombolini Riccardo , Woo Sheridan , Zeilinger Susanne , Lorito Matteo , Jansson Janet K.
Published: May 2004

Plant tissue colonization by
Trichoderma atroviride
plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between
gfp
-tagged biocontrol strains of
T. atroviride
and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of
T. atroviride
adhered to
Pythium ultimum
mycelia in coculture experiments. In mycoparasitic interactions of
T. atroviride
with
P. ultimum
or
Rhizoctonia solani
, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of
T. atroviride
mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of
P. ultimum
by
T. atroviride
occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific
Trichoderma
genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or
Rhizoctonia
by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.
Novel Forms of Structural Integration between Microbes and a Hydrothermal Vent Gastropod from the Indian Ocean
Authors: Goffredi Shana K. , Warén Anders , Orphan Victoria J. , Van Dover Cindy L. , Vrijenhoek Robert C.
Published: May 2004

Here we describe novel forms of structural integration between endo- and episymbiotic microbes and an unusual new species of snail from hydrothermal vents in the Indian Ocean. The snail houses a dense population of γ-proteobacteria within the cells of its greatly enlarged esophageal gland. This tissue setting differs from that of all other vent mollusks, which harbor sulfur-oxidizing endosymbionts in their gills. The significantly reduced digestive tract, the isotopic signatures of the snail tissues, and the presence of internal bacteria suggest a dependence on chemoautotrophy for nutrition. Most notably, this snail is unique in having a dense coat of mineralized scales covering the sides of its foot, a feature seen in no other living metazoan. The scales are coated with iron sulfides (pyrite and greigite) and heavily colonized by ε- and δ-proteobacteria, likely participating in mineralization of the sclerites. This novel metazoan-microbial collaboration illustrates the great potential of organismal adaptation in chemically and physically challenging deep-sea environments.
Vanadium Respiration by
Geobacter metallireducens
: Novel Strategy for In Situ Removal of Vanadium from Groundwater
Authors: Ortiz-Bernad Irene , Anderson Robert T. , Vrionis Helen A. , Lovley Derek R.
Published: May 2004

Vanadium can be an important contaminant in groundwaters impacted by mining activities. In order to determine if microorganisms of the
Geobacteraceae
, the predominant dissimilatory metal reducers in many subsurface environments, were capable of reducing vanadium(V),
Geobacter metallireducens
was inoculated into a medium in which acetate was the electron donor and vanadium(V) was the sole electron acceptor. Reduction of vanadium(V) resulted in the production of vanadium(IV), which subsequently precipitated. Reduction of vanadium(V) was associated with cell growth with a generation time of 15 h. No vanadium(V) was reduced and no precipitate was formed in heat-killed or abiotic controls. Acetate was the most effective of all the electron donors evaluated. When acetate was injected into the subsurface to enhance the growth and activity of
Geobacteraceae
in an aquifer contaminated with uranium and vanadium, vanadium was removed from the groundwater even more effectively than uranium. These studies demonstrate that
G. metallireducens
can grow via vanadium(V) respiration and that stimulating the activity of
Geobacteraceae
, and hence vanadium(V) reduction, can be an effective strategy for in situ immobilization of vanadium in contaminated subsurface environments.
Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants
Authors: Minamisawa Kiwamu , Nishioka Kiyo , Miyaki Taro , Ye Bin , Miyamoto Takuya , You Mu , Saito Asami , Saito Masanori , Barraquio Wilfredo L. , Teaumroong Neung , Sein Than , Sato Tadashi
Published: May 2004

We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N
2
-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N
2
fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N
2
fixation arises in association with nondiazotrophic endophytes.
Negative Cross-Communication among Wheat Rhizosphere Bacteria: Effect on Antibiotic Production by the Biological Control Bacterium
Pseudomonas aureofaciens
30-84
Authors: Morello J. E. , Pierson E. A. , Pierson L. S.
Published: May 2004

Phenazine antibiotic production in the biological control bacterium
Pseudomonas aureofaciens
30-84 is regulated in part via the PhzR/PhzI
N
-acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact.
11:
1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I
1
/I
2
. Growth of the
phzB
::
lacZ
reporter strain 30-84Z in this CM resulted in decreased
lacZ
expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of
phzB
occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of
phzR
and
phzI
, but not
phzI
alone, in
trans
into strain 30-84Z reduced the negative effect on
phzB
::
lacZ
expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.
Bifidobacterium lactisDSM 10140: Identification of theatp(atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny
Authors: Ventura Marco , Canchaya Carlos , van Sinderen Douwe , Fitzgerald Gerald F. , Zink Ralf
Published: May 2004

Theatpoperon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of theatpDgene to amplify partialatpDsequences from 12Bifidobacteriumspecies and nineLactobacillusspecies. All PCR products were sequenced and aligned with otheratpDsequences retrieved from public databases. Genes encoding the subunits of the F1F0-ATPase ofBifidobacterium lactisDSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genusBifidobacteriumand for the closely related taxaBifidobacterium lactisandBifidobacterium animalisandLactobacillus gasseriandLactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of theatpoperon, and a transcript at 4.5 kb, which corresponded to theatpC,atpD,atpG, andatpAgenes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of theatpDgenes demonstrated that theLactobacillus atpDgene clustered with the generaListeria,Lactococcus,Streptococcus, andEnterococcusand that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of theatpoperon ofB. lactisDSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures ofB. lactisDSM 10140. The rapid increase in the level ofatpoperon transcripts upon exposure to low pH suggested that the ATPase complex ofB. lactisDSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.