bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of
Bacillus subtilis
Encapsulated with Poly-γ-Glutamate
Authors: Kimura Keitarou , Itoh Yoshifumi
Published: May 2003

Some
Bacillus subtilis
strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in
B. subtilis
host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn
2+
or Mn
2+
ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding
pghP
gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated
M
r
of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in
B. subtilis
cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.
Cloning and Sequencing of a Poly(
dl
-Lactic Acid) Depolymerase Gene from
Paenibacillus amylolyticus
Strain TB-13 and Its Functional Expression in
Escherichia coli
Authors: Akutsu-Shigeno Yukie , Teeraphatpornchai Teerawat , Teamtisong Kamonluck , Nomura Nobuhiko , Uchiyama Hiroo , Nakahara Tadaatsu , Nakajima-Kambe Toshiaki
Published: May 2003

The gene encoding a poly(
dl-
lactic acid) (PLA) depolymerase from
Paenibacillus amylolyticus
strain TB-13 was cloned and overexpressed in
Escherichia coli
. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-
co
-adipate), poly(ethylene succinate), and poly(ε-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and
p
-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic
Bacillus
species. The identity of the amino acid sequence of PlaA with
Bacillus
lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.
Cell Culture-Taqman PCR Assay for Evaluation of
Cryptosporidium parvum
Disinfection
Authors: Keegan Alexandra R. , Fanok Stella , Monis Paul T. , Saint Christopher P.
Published: May 2003

Cryptosporidium parvum
represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of
C. parvum
with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log
10
units) with LP-UV (20 mJ/cm
2
) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log
10
unit being inactivated. These results demonstrate the inability of MIOX to inactivate
Cryptosporidium
. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.
TheftsHGene of the Wine BacteriumOenococcus oeniIs Involved in Protection against Environmental Stress
Authors: Bourdineaud Jean-Paul , Nehmé Benjamin , Tesse Sonia , Lonvaud-Funel Aline
Published: May 2003

The wine bacteriumOenococcus oenihas to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of theO. oeni ftsHgene, encoding a protease belonging to the ATP binding cassette protein superfamily. TheO. oeniFtsH protein is closest in sequence similarity to the FtsH homologue ofLactococcus lactis. TheO. oeni ftsHgene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock.O. oeniFtsH protein function was tested in anEscherichia coli ftsHmutant strain, and consistent with theO. oeni ftsHgene expression pattern, theO. oeniFtsH protein provided protection for theE. coli ftsHmutant against heat shock.O. oeniandBradyrhizobium japonicumFtsH proteins also triggeredE. coliresistance to wine toxicity. Genes homologous toO. oeni ftsHwere detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.
Global Expression Profiling and Physiological Characterization ofCorynebacterium glutamicumGrown in the Presence ofl-Valine
Authors: Lange C. , Rittmann D. , Wendisch V. F. , Bott M. , Sahm H.
Published: May 2003

Addition ofl-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-typeCorynebacterium glutamicumATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvAΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increasedilvBNmRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of theilvBprotein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations ofl-isoleucine could relieve the valine effect on VAL1 whereasl-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to theilvAdeletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression ofilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.
Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (
opd
) Gene Cluster Found in
Flavobacterium
sp
Authors: Siddavattam Dayananda , Khajamohiddin Syed , Manavathi Bramanandam , Pakala Suresh B. , Merrick Mike
Published: May 2003

Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (
opd
) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the
opd
gene is highly conserved. Two
opd
plasmids, pPDL2 from
Flavobacterium
sp. and pCMS1 from
Pseudomonas diminuta
, are well characterized, and in each of them a region of about 5.1 kb containing the
opd
gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The
opd
gene is flanked upstream by an insertion sequence, ISFlsp
1
, that is a member of the IS
21
family, and downstream by a Tn
3
-like element encoding a transposase and a resolvase. Adjacent to
opd
but transcribed in the opposite direction is an open reading frame (
orf243
) with the potential to encode an aromatic hydrolase somewhat similar to
Pseudomonas putida
TodF. We have shown that
orf243
encodes a polypeptide of 27 kDa, which plays a role in the degradation of
p
-nitrophenol and is likely to act in concert with
opd
in the degradation of parathion. The linkage of
opd
and
orf243
, the organization of the genes flanking
opd
, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.
Factors Influencing Survival of
Legionella pneumophila
Serotype 1 in Hot Spring Water and Tap Water
Authors: Ohno Akira , Kato Naoyuki , Yamada Koji , Yamaguchi Keizo
Published: May 2003

The factors involved in the survival of
Legionella pneumophila
in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity.
L. pneumophila
could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42°C, except for one microcosm with a pH of <2.0. Lower temperatures supported survival without loss of cultivability. The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45°C. The optimal range of pH for survival was between 6.0 and 8. The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt. The cultivability of organisms in the post-exponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase. In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase. Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected. Resuscitation by the addition of
Acanthamoeba castellanii
to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population. Our results suggest that
L. pneumophila
cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas.
Determining Diversity of Freshwater Fungi on Decaying Leaves: Comparison of Traditional and Molecular Approaches
Authors: Nikolcheva Liliya G. , Cockshutt Amanda M. , Bärlocher Felix
Published: May 2003

Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of
Articulospora tetracladia
dominated. Modifications of two molecular methods—denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis—suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of
A. tetracladia
. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams.
Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure
Authors: Egert Markus , Friedrich Michael W.
Published: May 2003

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using
Msp
I and
Alu
I as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.
Enhanced Killing of
Acanthamoeba
Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System
Authors: Hughes Reanne , Andrew Peter W. , Kilvington Simon
Published: May 2003

The activity of H
2
O
2
against the resistant cyst stage of the pathogenic free-living amoeba
Acanthamoeba
was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H
2
O
2
, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (
P
< 0.05). With 2% H
2
O
2
, enhancement was observed at all time points (
P
< 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H
2
O
2
was enhanced to give 3-log killing after 8 h of exposure (
P
< 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H
2
O
2
was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H
2
O
2
in contact lens care systems, the enhanced 2% H
2
O
2
system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H
2
O
2
occurred by 4 h. In contrast, 2% H
2
O
2
alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H
2
O
2
contact lens disinfection systems against
Acanthamoeba
cysts and prevention of acanthamoeba keratitis.
Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish
Authors: Takahashi Hajime , Kimura Bon , Yoshikawa Miwako , Fujii Tateo
Published: May 2003

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (
hdc
) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of
Citrobacter braakii
. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial
hdc
was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of
hdc
was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.
Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate
Heterocapsa circularisquama
Authors: Nagasaki Keizo , Tomaru Yuji , Tarutani Kenji , Katanozaka Noriaki , Yamanaka Satoshi , Tanabe Hiroshi , Yamaguchi Mineo
Published: May 2003

The growth characteristics and intraspecies host specificity of
Heterocapsa circularisquama
virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate
H. circularisquama
, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to
Heterosigma akashiwo
virus, HcV was more widely infectious to
H. circularisquama
strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.
Genetic Characterization of
Escherichia coli
Populations from Host Sources of Fecal Pollution by Using DNA Fingerprinting
Authors: McLellan Sandra L. , Daniels Annette D. , Salmore Alissa K.
Published: May 2003

Escherichia coli
isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (
n
= 180), gulls (
n
= 133), and dairy cattle (
n
= 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of
E. coli
fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of
E. coli
contamination will require extensive isolation of strains to encompass
E. coli
strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.
Insertional Mutagenesis of a Fungal Biocontrol Agent Led to Discovery of a Rare Cellobiose Lipid with Antifungal Activity
Authors: Cheng Yali , McNally David J. , Labbé Caroline , Voyer Normand , Belzile François , Bélanger Richard R.
Published: May 2003

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent,
Pseudozyma flocculosa
, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2′,4′-diacetoxy-5′-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of
P. flocculosa
.
A Novel NAD-Dependent Dehydrogenase, Highly Specific for 1,5-Anhydro-
d
-Glucitol, from
Trichoderma longibrachiatum
Strain 11-3
Authors: Yoshida Nobuyuki , Uchida Etsuko , Katsuragi Tohoru , Tani Yoshiki
Published: May 2003

A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-
d
-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus,
Trichoderma longibrachiatum
strain 11-3. This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose. 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric. The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG. A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed. The presence of glucose in abundance did not interfere with the relationship. The optimum temperature for the enzyme reaction was 50°C, and the enzyme was stable at temperatures up to 70°C. These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus.
An Exocellular Protein from the Oil-Degrading Microbe
Acinetobacter venetianus
RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan
Authors: Bach Horacio , Berdichevsky Yevgeny , Gutnick David
Published: May 2003

The oil-degrading microorganism
Acinetobacter venetianus
RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as
n
-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in
Escherichia coli
BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in
E. coli.
Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism
Acinetobacter calcoaceticus
BD4, showed no enhancement.
Naturally Occurring Bacteria Similar to the Methyl
tert
-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater
Authors: Hristova Krassimira , Gebreyesus Binyam , Mackay Douglas , Scow Kate M.
Published: May 2003

Methyl
tert
-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by
Taq
Man quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10
3
to 10
4
cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10
5
cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.
Are Readily Culturable Bacteria in Coastal North Sea Waters Suppressed by Selective Grazing Mortality?
Authors: Beardsley Christine , Pernthaler Jakob , Wosniok Werner , Amann Rudolf
Published: May 2003

We studied the growth of six culturable bacterial lineages from coastal North Sea picoplankton in environmental samples under different incubation conditions. The grazing pressure of heterotrophic nanoflagellates (HNF) was reduced either by double prefiltration through 0.8-μm-pore-size filters or by 10-fold dilutions with 0.2-μm (pore-size) prefiltered seawater. We hypothesized that those γ-proteobacterial genera that are rapidly enriched would also be most strongly affected by HNF regrowth. In the absence of HNF, the mean protein content per bacterial cell increased in both treatments compared to environmental samples, whereas the opposite trend was found in incubations of unaltered seawater. Significant responses to the experimental manipulations were observed in
Alteromonas
,
Pseudoalteromonas
, and
Vibrio
populations. No treatment-specific effects could be detected for members of the
Roseobacter
group, the
Cytophaga latercula-C. marinoflava
lineage, or the NOR5 clade. Statistical analysis confirmed a transient increase in the proportions of
Alteromonas
,
Pseudoalteromonas
, and
Vibrio
cells at reduced HNF densities only, followed by an overproportional decline during the phase of HNF regrowth. Cells from these genera were significantly larger than the community average in the dilution treatments, and changes in their relative abundances were negatively correlated with HNF densities. Our findings suggest that bacteria affiliated with frequently isolated genera such as
Alteromonas
,
Pseudoalteromonas
, and
Vibrio
might be rare in coastal North Sea picoplankton because their rapid growth response to changing environmental conditions is counterbalanced by a higher grazing mortality.
Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization
Authors: Pernthaler Jakob , Pernthaler Annelie , Amann Rudolf
Published: May 2003

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized
z
-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.
Genetic Organization and Molecular Analysis of the
Eco
VIII Restriction-Modification System of
Escherichia coli
E1585-68 and Its Comparison with Isospecific Homologs
Authors: Mruk Iwona , Kaczorowski Tadeusz
Published: May 2003

The
Eco
VIII restriction-modification (R-M) system is carried by the
Escherichia coli
E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the
Eco
VIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or
E. coli
genomic DNA (50.8%). The difference suggests that there is a possibility that the
Eco
VIII R-M system was recently acquired by the genome. The 921-bp
Eco
VIII endonuclease (R ·
Eco
VIII) gene (
ecoVIIIR
) encodes a 307-amino-acid protein with an
M
r
of 35,554. The convergently oriented
Eco
VIII methyltransferase (M ·
Eco
VIII) gene (
ecoVIIIM
) consists of 912 bp that code for a 304-amino-acid protein with an
M
r
of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of
Eco
VIII R-M enzymes purified to homogeneity were characterized. R ·
Eco
VIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M ·
Eco
VIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to
N
6
-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the
Hin
dIII (
Haemophilus influenzae
Rd) and
Lla
CI (
Lactococcus lactis
subsp.
cremoris
W15) R-M systems. This finding is reflected by the levels of homology of M ·
Eco
VIII with M ·
Hin
dIII and M ·
Lla
CI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m
6
N
-adenine β-class methyltransferases. The deduced amino acid sequence of R ·
Eco
VIII shows weak homology with its two isoschizomers, R ·
Hin
dIII (26%) and R ·
Lla
CI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R ·
Eco
VIII (D
108
X
12
DXK
123
), as well as in the primary structures of R ·
Lla
CI and R ·
Hin
dIII. Polyclonal antibodies raised against R ·
Eco
VIII did not react with R ·
Hin
dIII, while anti-M ·
Eco
VIII antibodies cross-reacted with M ·
Lla
CI but not with M ·
Hin
dIII. R ·
Eco
VIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M ·
Eco
VIII enzyme. The biological implications of this finding are discussed.
Use of
Bifidobacterium dentium
as an Indicator of the Origin of Fecal Water Pollution
Authors: Nebra Yolanda , Bonjoch Xavier , Blanch Anicet R.
Published: May 2003

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain
Bifidobacterium
species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of
Bifidobacterium dentium
, a
Bifidobacterium
species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several
Bifidobacterium
species related with animal origins. The specificity of both probes was evaluated by using 24
Bifidobacterium
species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for
B. dentium
, whereas that with the BAN probe showed it to be specific for
B. animalis
,
B. asteroides
,
B. coryneforme
,
B. cuniculi
,
B. globosum
,
B. magnum
,
B. minimum
, and
B. subtile.
A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.
Novel Eukaryotic Lineages Inferred from Small-Subunit rRNA Analyses of Oxygen-Depleted Marine Environments
Authors: Stoeck Thorsten , Epstein Slava
Published: May 2003

Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.
Large-Scale Spatial Distribution of Virioplankton in the Adriatic Sea: Testing the Trophic State Control Hypothesis
Authors: Corinaldesi C. , Crevatin E. , Del Negro P. , Marini M. , Russo A. , Fonda-Umani S. , Danovaro R.
Published: May 2003

Little is known concerning environmental factors that may control the distribution of virioplankton on large spatial scales. In previous studies workers reported high viral levels in eutrophic systems and suggested that the trophic state is a possible driving force controlling the spatial distribution of viruses. In order to test this hypothesis, we determined the distribution of viral abundance and bacterial abundance and the virus-to-bacterium ratio in a wide area covering the entire Adriatic basin (Mediterranean Sea). To gather additional information on factors controlling viral distribution on a large scale, functional microbial parameters (exoenzymatic activities, bacterial production and turnover) were related to trophic gradients. At large spatial scales, viral distribution was independent of autotrophic biomass and all other environmental parameters. We concluded that in contrast to what was previously hypothesized, changing trophic conditions do not directly affect virioplankton distribution. Since virus distribution was coupled with bacterial turnover times, our results suggest that viral abundance depends on bacterial activity and on host cell abundance.
Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in
Lactobacillus plantarum
,
L. paraplantarum, L. pentosus
, and
L. casei
: Prevalence of CO
2
-Dependent Auxotrophs and Characterization of Deficient
arg
Genes in
L. plantarum
Authors: Bringel Françoise , Hubert Jean-Claude
Published: May 2003

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO
2
. We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of
Lactobacillus plantarum
, 32 strains of
L. pentosus
, 15 strains of
L. paraplantarum
, and 10 strains of
L. casei
. The distribution of prototroph and auxotroph phenotypes varied between species. All
L. casei
strains, no
L. paraplantarum
strains, two
L. pentosus
strains, and seven
L. plantarum
strains required arginine for growth. Arginine auxotrophs were more frequently found in
L. plantarum
isolated from milk products than in
L. plantarum
isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In
L. plantarum
the
argCJBDF
genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different
arg
genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one
L. pentosus
strain, a high CO
2
requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207
Lactobacillus
strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.
A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library
Authors: Liles Mark R. , Manske Brian F. , Bintrim Scott B. , Handelsman Jo , Goodman Robert M.
Published: May 2003

We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the
Bacillus
, α-
Proteobacteria
, and
CFB
groups; greater representation among the β- and γ-
Proteobacteria
, and
OP10
divisions; and no rRNA genes from the domains
Eukaryota
and
Archaea
. In addition to rRNA genes recovered from the bacterial divisions
Proteobacteria
,
Verrucomicrobia
,
Firmicutes
,
Cytophagales
, and
OP11
, we identified many rRNA genes from the BAC library affiliated with the bacterial division
Acidobacterium
; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the
Acidobacterium
division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the
Acidobacterium
division.
Role of
Listeria monocytogenes
σ
B
in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response
Authors: Ferreira Adriana , Sue David , O'Byrne Conor P. , Boor Kathryn J.
Published: May 2003

The food-borne pathogen
Listeria monocytogenes
can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ
B
, which is encoded by
sigB
, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in
L. monocytogenes
. At various points throughout growth, we compared the relative survival of
L. monocytogenes
wild-type and Δ
sigB
strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the Δ
sigB
strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both Δ
sigB
and wild-type
L. monocytogenes
strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the
L. monocytogenes
growth phase-dependent AR mechanism is σ
B
independent. σ
B
-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ
B
reduced the survival of
L. monocytogenes
at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that
L. monocytogenes
protection against organic and inorganic acid may be mediated through different mechanisms. σ
B
does not appear to contribute to pH
i
homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH
i
buffering by the GAD system under the conditions examined in this study. In summary, a functional σ
B
protein is necessary for full resistance of
L. monocytogenes
to lethal acid treatments.
Production of Plant-Specific Flavanones by
Escherichia coli
Containing an Artificial Gene Cluster
Authors: Hwang Eui Il , Kaneko Masafumi , Ohnishi Yasuo , Horinouchi Sueharu
Published: May 2003

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in
Escherichia coli
, three sets of an artificial gene cluster which contained three genes of heterologous origins—
PAL
from the yeast
Rhodotorula rubra
,
4CL
from the actinomycete
Streptomyces coelicolor
A3(2), and
CHS
from the licorice plant
Glycyrrhiza echinata—
were constructed. The constructions of the three sets were done as follows: (i)
PAL
,
4CL
, and
CHS
were placed in that order under the control of the T7 promoter (P
T7
) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of
4CL
and
CHS
were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P
T7
in front of
PAL
, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P
T7
and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid.
E. coli
cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.