bluuebunny/crossref_metadata_2025 huggingface dataset:
A Homologue of the Tryptophan-Rich Sensory Protein TspO and FixL Regulate a Novel Nutrient Deprivation-Induced
Sinorhizobium meliloti
Locus
Authors: Davey Mary Ellen , de Bruijn Frans J.
Published: July 2002

A nutrient deprivation-induced locus in
Sinorhizobium meliloti
strain 1021 was identified by use of a Tn
5-luxAB
reporter gene transposon. The tagged locus is comprised of two open reading frames (ORFs) designated
ndiA
and
ndiB
for nutrient deprivation-induced genes A and B. Comparison of the deduced amino acid sequences of both
ndiA
and
ndiB
to the protein databases failed to reveal similarity to any known genes. The expression of the
ndi
locus was found to be induced by carbon and nitrogen deprivation, osmotic stress, and oxygen limitation and during entry into stationary phase. To identify regulatory components involved in the control of
ndi
gene expression, a second round of mutagenesis was performed on the primary
ndiB
::Tn
5-luxAB
-tagged strain (C22) with transposon Tn
1721
. A double-mutant strain was obtained that lacked
ndi
locus transcriptional activity under all of the inducing conditions tested. The Tn
1721
-tagged gene showed a high degree of similarity to tryptophan-rich sensory protein TspO from
Rhodobacter sphaeroides
, as well as to mitochondrial benzodiazepine receptor pK18 from mammals. Induction of the
ndi
::Tn
5-luxAB
reporter gene fusion was restored under all inducing conditions by introducing the
tspO
coding region, from either
S. meliloti
or
R. sphaeroides
, in
trans
. Furthermore, it was found that, in addition to
tspO
,
fixL
, which encodes the sensor protein of an oxygen-sensing two-component system, is required for full expression of the
ndi
locus, but only under low oxygen tension.
Expression of Outer Membrane Proteins in
Escherichia coli
Growing at Acid pH
Authors: Sato Mikiko , Machida Kazuhiro , Arikado Eri , Saito Hiromi , Kakegawa Tomohito , Kobayashi Hiroshi
Published: July 2002

It is generally accepted for
Escherichia coli
that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of
ompF
at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an
ompR
mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant
envZ
strain, and their expression was regulated by medium osmolarity. Thus, it appears that
E. coli
has a different mechanism for porin expression at acid pH. A mutant deficient in
ompR
grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when
ompC
and
ompF
were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.
Increase in Bacterial Community Diversity in Subsurface Aquifers Receiving Livestock Wastewater Input
Authors: Cho Jang-Cheon , Kim Sang-Jong
Published: July 2002

Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the
Cytophaga-Flexibacter-Bacteroides
and low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock.
Evaluation of F-Specific RNA Bacteriophage as a Candidate Human Enteric Virus Indicator for Bivalve Molluscan Shellfish
Authors: Doré William J. , Henshilwood Kathleen , Lees David N.
Published: July 2002

Escherichia coli
is a widely utilized indicator of the sanitary quality of bivalve molluscan shellfish sold for human consumption. However, it is now well documented that shellfish that meet the
E. coli
standards for human consumption may contain human enteric viruses that cause gastroenteritis and hepatitis. In this study we investigated using F-specific RNA bacteriophage (FRNA bacteriophage) to indicate the likely presence of such viruses in shellfish sold for consumption. FRNA bacteriophage and
E. coli
levels were determined over a 2-year period for oysters (
Crassostrea gigas
) harvested from four commercial sites chosen to represent various degrees of sewage pollution. Three sites were classified as category B sites under the relevant European Community (EC) Directive (91/492), which required purification (depuration) of oysters from these sites before sale. One site was classified as a category A site, and oysters from this site could be sold directly without further processing. Samples were tested at the point of sale following commercial processing and packaging. All of the shellfish complied with the mandatory EC
E. coli
standard (less than 230 per 100 g of shellfish flesh), and the levels of contamination for more than 90% of the shellfish were at or below the level of sensitivity of the assay (20
E. coli
MPN per 100 g), which indicated good quality based on this criterion. In contrast, FRNA bacteriophage were frequently detected at levels that exceeded 1,000 PFU per 100 g. High levels of FRNA bacteriophage contamination were strongly associated with harvest area fecal pollution and with shellfish-associated disease outbreaks. Interestingly, FRNA bacteriophage contamination exhibited a marked seasonal trend that was consistent with the trend of oyster-associated gastroenteritis in the United Kingdom. The correlation between FRNA bacteriophage contamination and health risk was investigated further by using a reverse transcription-PCR assay for Norwalk-like virus (NLV). NLV contamination of oysters was detected only at the most polluted site and also exhibited a seasonal trend that was consistent with the trend of FRNA bacteriophage contamination and with the incidence of disease. The results of this study suggest that FRNA bacteriophage could be used as viral indicators for market-ready oysters.
Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by
Enterococcus faecalis
BFE 1071
Authors: Balla E. , Dicks L. M. T. , Du Toit M. , Van Der Merwe M. J. , Holzapfel W. H.
Published: July 2002

The pH-neutral cell supernatant of
Enterococcus faecalis
BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of
Enterococcus
spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to
E. faecalis
strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames,
ent
1071A and
ent
1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of
E. faecalis
bacteriocin.
The Viable-but-Nonculturable State Induced by Abiotic Stress in the Biocontrol Agent
Pseudomonas fluorescens
CHA0 Does Not Promote Strain Persistence in Soil
Authors: Mascher Fabio , Hase Carsten , Moënne-Loccoz Yvan , Défago Geneviève
Published: July 2002

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant
Pseudomonas fluorescens
CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the waterlogged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.
Differential Damage in Bacterial Cells by Microwave Radiation on the Basis of Cell Wall Structure
Authors: Woo Im-Sun , Rhee In-Koo , Park Heui-Dong
Published: July 2002

Microwave radiation in
Escherichia coli
and
Bacillus subtilis
cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most
E. coli
cells, yet there was no significant change observed in the
B. subtilis
cells. Microwave-injured
E. coli
cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet
B. subtilis
cells were resistant to SDS.
Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System
Authors: Whiteley Andrew S. , Bailey Mark J.
Published: July 2002

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the α, β and γ subclasses of the class
Proteobacteria
[α-, β-, and γ-
Proteobacteria
, respectively], the
Cytophaga-Flavobacterium
group, and the
Pseudomonas
group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the
Cytophaga-Flavobacterium
group and to the γ-
Proteobacteria
. The α subclass
Proteobacteria
were of low relative abundance throughout the treatment system whilst the β subclass of the
Proteobacteria
exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The γ subclass of the
Proteobacteria
had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the
Cytophaga-Flavobacterium
group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the γ-
Proteobacteria
and the
Cytophaga-Flavobacteria
during this bioremediation process was highlighted.
Antibiosis between Ruminal Bacteria and Ruminal Fungi
Authors: Dehority Burk A. , Tirabasso Patricia A.
Published: July 2002

Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (
P
< 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.
Culturability and In Situ Abundance of Pelagic Bacteria from the North Sea
Authors: Eilers Heike , Pernthaler Jakob , Glöckner Frank Oliver , Amann Rudolf
Published: July 2002

The culturability of abundant members of the domain
Bacteria
in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera
Pseudoalteromonas
,
Alteromonas
, and
Vibrio
. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the
Cytophaga-Flavobacteria
and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the
Cytophaga-Flavobacterum
cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus
Roseobacter
. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.
Colonization of Corn,
Zea mays
, by the Entomopathogenic Fungus
Beauveria bassiana
Authors: Wagner Bruce L. , Lewis Leslie C.
Published: July 2002

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus
Beauveria bassiana
(Balsamo) Vuillemin into corn,
Zea mays
L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that
B. bassiana
does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how
B. bassiana
travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that
B. bassiana
does not lose virulence toward the European corn borer,
Ostrinia nubilalis
(Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.
Microbial Community Dynamics during Production of the Mexican Fermented Maize Dough Pozol
Authors: ben Omar Nabil , Ampe Frédéric
Published: July 2002

The dynamics of the microbial community responsible for the traditional fermentation of maize in the production of Mexican pozol was investigated by using a polyphasic approach combining (i) microbial enumerations with culture media, (ii) denaturing gradient gel electrophoresis (DGGE) fingerprinting of total community DNA with bacterial and eukaryotic primers and sequencing of partial 16S ribosomal DNA (rDNA) genes, (iii) quantification of rRNAs from dominant microbial taxa by using phylogenetic oligonucleotide probes, and (iv) analysis of sugars and fermentation products. A
Streptococcus
species dominated the fermentation and accounted for between 25 and 75% of the total flora throughout the process. Results also showed that the initial epiphytic aerobic microflora was replaced in the first 2 days by heterofermentative lactic acid bacteria (LAB), including a close relative of
Lactobacillus fermentum
, producing lactic acid and ethanol; this heterolactic flora was then progressively replaced by homofermentative LAB (mainly close relatives of
L. plantarum
,
L. casei
, and
L. delbrueckii
) which continued acidification of the maize dough. At the same time, a very diverse community of yeasts and fungi developed, mainly at the periphery of the dough. The analysis of the DGGE patterns obtained with bacterial and eukaryotic primers targeting the 16S and 18S rDNA genes clearly demonstrated that there was a major shift in the community structure after 24 h and that high biodiversity—according to the Shannon-Weaver index—was maintained throughout the process. These results proved that a relatively high number of species, at least six to eight, are needed to perform this traditional lactic acid fermentation. The presence of
Bifidobacterium
,
Enterococcus
, and enterobacteria suggests a fecal origin of some important pozol microorganisms. Overall, the results obtained with different culture-dependent or -independent techniques clearly confirmed the importance of developing a polyphasic approach to study the ecology of fermented foods.
Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria
Authors: Spear John R. , Figueroa Linda A. , Honeyman Bruce D.
Published: July 2002

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB
Desulfovibrio vulgaris
along with a
Clostridium
sp. determined via 16S ribosomal DNA analysis. The pure culture was
Desulfovibrio desulfuricans
(ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate,
V
max
, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h
−1
) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h
−1
at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture,
V
max
was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h
−1
at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min
−1
for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min
−1
(U
0
= 1 mM) for the
D. desulfuricans
culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.
Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by
Lactobacillus delbrueckii
subsp.
bulgaricus
SS1 and
Lactococcus lactis
subsp.
cremoris
FT4
Authors: Gobbetti M. , Ferranti P. , Smacchi E. , Goffredi F. , Addeo F.
Published: July 2002

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected
Lactobacillus delbrueckii
subsp.
bulgaricus
SS1 and
L. lactis
subsp.
cremoris
FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by
L. delbrueckii
subsp.
bulgaricus
SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by
L. lactis
subsp.
cremoris
FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC
50
s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC
50
s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.
Engineering Hydrogen Sulfide Production and Cadmium Removal by Expression of the Thiosulfate Reductase Gene (
phsABC
) from
Salmonella enterica
Serovar Typhimurium in
Escherichia coli
Authors: Bang Sang-Weon , Clark Douglas S. , Keasling Jay D.
Published: July 2002

The thiosulfate reductase gene (
phsABC
) from
Salmonella enterica
serovar Typhimurium was expressed in
Escherichia coli
to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing
phsABC
was inserted into the pMB1-based, high-copy, isopropyl-β-
d
-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy,
m
-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3.7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107.
E. coli
DH5α strains harboring the
phsABC
constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four
phsABC
constructs,
E. coli
DH5α (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 μM and 91% of 200 μM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.
Identification and Characteristics of a Novel
Burkholderia
Strain with Broad-Spectrum Antimicrobial Activity
Authors: Cain Cody C. , Henry Alexis T. , Waldo Robert H. , Casida Lester J. , Falkinham Joseph O.
Published: July 2002

A
Burkholderia
strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.
Lactobacillus coryniformis
subsp.
coryniformis
Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound
Authors: Magnusson Jesper , Schnürer Johan
Published: July 2002

The antifungal activity spectrum of
Lactobacillus coryniformis
subsp.
coryniformis
strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds
Aspergillus fumigatus
,
A. nidulans
,
Penicillium roqueforti
,
Mucor hiemalis
,
Talaromyces flavus
,
Fusarium poae
,
F. graminearum
,
F. culmorum
, and
F. sporotrichoides
. A weaker activity was observed against the yeasts
Debaryomyces hansenii
,
Kluyveromyces marxianus
, and
Saccharomyces cerevisiae
. The yeasts
Rhodotorula glutinis
,
Sporobolomyces roseus
, and
Pichia anomala
were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121°C for 15 min. Maximum activity was observed at pH values of between 3.0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH
4
)
2
SO
4
precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from
L. coryniformis
subsp.
coryniformis
.
Mutation of the
ptsG
Gene Results in Increased Production of Succinate in Fermentation of Glucose by
Escherichia coli
Authors: Chatterjee Ranjini , Millard Cynthia Sanville , Champion Kathleen , Clark David P. , Donnelly Mark I.
Published: July 2002

Escherichia coli
NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the
ptsG
gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB
glc
. Replacement of the chromosomal
ptsG
gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the
ptsG
gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of
E. coli
, including
E. coli
B.
Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy
Authors: Auty M. A. E. , Gardiner G. E. , McBrearty S. J. , O'Sullivan E. O. , Mulvihill D. M. , Collins J. K. , Fitzgerald G. F. , Stanton C. , Ross R. P.
Published: July 2002

The viability of the human probiotic strains
Lactobacillus paracasei
NFBC 338 and
Bifidobacterium
sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD
Bac
Light viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD
Bac
Light staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD
Bac
Light staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ∼10
8
bacteria/ml (equivalent to ∼10
7
CFU/ml), based on
Bifidobacterium
sp. strain UCC 35612 counts in maximum-recovery diluent.
Chromosomal Locus for Cadmium Resistance in
Pseudomonas putida
Consisting of a Cadmium-Transporting ATPase and a MerR Family Response Regulator
Authors: Lee Seon-Woo , Glickmann Eric , Cooksey Donald A.
Published: July 2002

Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharactarized. A chromosomal fragment encoding cadmium resistance was cloned from
Pseudomonas putida
06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes,
cadA
and
cadR
. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from
Escherichia coli
. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene,
zntR
, regulates
zntA
in
E. coli
, but it is not contiguous with
zntA
in the
E. coli
genome as
cadA
and
cadR
were in
P. putida
. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the
P. aeruginosa
genome, the
P. putida
CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of
P. aeruginosa
also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that
cadA
and
cadR
are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike
zntA
, they did not confer significant levels of lead resistance. The
cadA
promoter was responsive to Cd(II), Pb(II), and Zn(II), while the
cadR
promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress
cadA
. Homologs of the cadmium-transporting ATPase were detected in many other
Pseudomonas
species.
Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil
Authors: Mishra Sanjeet , Jyot Jeevan , Kuhad Ramesh C. , Lal Banwari
Published: July 2002

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10
3
to 10
4
CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced
Acinetobacter baumannii
strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.
Enhanced Acid Sensitivity of Pressure-Damaged
Escherichia coli
O157 Cells
Authors: Pagán Rafael , Jordan Sarah , Benito Amparo , Mackey Bernard
Published: July 2002

Pressure-damaged
Escherichia coli
O157 cells were more acid sensitive than native cells and were impaired in pH homeostasis. However differences in acid sensitivity were not related to differences in cytoplasmic pH (pH
i
). Cellular β-galactosidase was more acid labile in damaged cells. Sensitization to acid may thus involve loss of protective or repair functions.
Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation ofNitrosomonasandNitrosospiraStrains
Authors: Brandt Kristian K. , Hesselso/e Martin , Roslev Peter , Henriksen Kaj , So/rensen Jan
Published: July 2002

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. TwoNitrosospirastrains were considerably more sensitive to LAS than twoNitrosomonasstrains were. Interestingly, the twoNitrosospirastrains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC50], 6 to 38 mg liter−1) was affected much less by LAS than the growth rate and viability (EC50, 3 to 14 mg liter−1) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated withNitrosomonas europaeagrown at a sublethal LAS level (10 mg liter−1); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH4+oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.
Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of
Staphylococcus aureus
Using Micro-Representational-Difference Analysis
Authors: Becker Petra , Hufnagle Wendy , Peters Georg , Herrmann Mathias
Published: July 2002

Microbial proliferation and biofilm formation on biologic or inert substrates are characteristics of invasive
Staphylococcus aureus
infections and is associated with phenotypic alterations such as reduced antimicrobial susceptibility. To identify genes which are typically expressed in biofilms, a micro-representational-difference analysis (micro-RDA) was adapted for gram-positive bacteria and used with cDNA derived from populations of
S. aureus
DSM 20231 growing in a biofilm or plankonically. In comparison to previously described cDNA RDA protocols, micro-RDA has the advantages that only minimal quantities of total RNA are needed and, most importantly, that total RNA can be used since the large amount of rRNA in total RNA does not interfere with the micro-RDA procedure. Using a series of spiked controls with various amounts of MS2 RNA in a background of total RNA from
S. aureus,
the equivalent of five copies of MS2 per cell were detectable after three rounds of subtractive enrichment. Five genes were identified as being differentially expressed in biofilm versus planktonic cultures. These genes revealed homology to a threonyl-tRNA synthetase, a phosphoglycerate mutase, a triosephosphate isomerase, an alcohol dehydrogenase I, and a ClpC ATPase. Differential levels of expression were subsequently confirmed by standard Northern blotting. In conclusion, micro-RDA is a sensitive and specific method to detect transcripts differentially expressed as a function of different
S. aureus
growth conditions.
Microbial Diversity of the Brine-Seawater Interface of the Kebrit Deep, Red Sea, Studied via 16S rRNA Gene Sequences and Cultivation Methods
Authors: Eder Wolfgang , Jahnke Linda L. , Schmidt Mark , Huber Robert
Published: July 2002

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus
Halanaerobium
and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus
Halanaerobium
, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other
Halanaerobium
representatives, showing unusually large amounts of Δ7 and Δ11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus
Halanaerobium
. The new
Halanaerobium
16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.
Nuclear Magnetic Resonance Analysis of [1-
13
C]Dimethylsulfoniopropionate (DMSP) and [1-
13
C]Acrylate Metabolism by a DMSP Lyase-Producing Marine Isolate of the α-Subclass of
Proteobacteria
Authors: Ansede John H. , Pellechia Perry J. , Yoch Duane C.
Published: July 2002

The prominence of the α-subclass of
Proteobacteria
in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-
13
C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-
13
C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-
13
C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-
13
C]DMSP and [1-
13
C]β-hydroxypropionate ([1-
13
C]β-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly β-hydroxylated upon formation. When [1-
13
C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-
13
C]β-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was β-hydroxylated on (or near) the cell surface to β-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and β-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the α-proteobacterium, strain LFR.
Development of a Genetic System for
Geobacter sulfurreducens
Authors: Coppi Maddalena V. , Leang Ching , Sandler Steven J. , Lovley Derek R.
Published: July 2002

Members of the genus
Geobacter
are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for
Geobacter sulfurreducens
. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into
G. sulfurreducens
by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in
G. sulfurreducens.
In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the
nifD
gene of
G. sulfurreducens
was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of
G. sulfurreducens
to grow in medium lacking a source of fixed nitrogen. Expression of the
nifD
gene in
trans
complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the
Geobacter
genus.
Effects of Cell-Bound Microcystins on Survival and Feeding of
Daphnia
spp
Authors: Rohrlack Thomas , Dittmann Elke , Börner Thomas , Christoffersen Kirsten
Published: July 2002

The influence of cell-bound microcystins on the survival time and feeding rates of six
Daphnia
clones belonging to five common species was studied. To do this, the effects of the microcystin-producing
Microcystis
strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the
Daphnia
clones and
Daphnia
survival time was analyzed. Microcystins ingested with
Microcystis
cells were poisonous to all
Daphnia
clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among
Daphnia
clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of
Daphnia
. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for
Daphnia
spp., (ii)
Daphnia
spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii)
Daphnia
spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.