bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTMicroeukaryotes 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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.
Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from
Arthrobacter
sp. Strain SU
Authors: Zhuang Zhihao , Gartemann Karl-Heinz , Eichenlaub Rudolf , Dunaway-Mariano Debra 
Published: May 2003

ABSTRACT

The
Arthrobacter
sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes
fcbA
,
fcbB
, and
fcbC
(A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes
fcbA
and
fcbB
encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of
fcbC
is not known. We subcloned
fcbC
and expressed it in
Escherichia coli
, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a
k
cat
of 6.7 s
−1
and a

K
m

of 1.2 μM. The
k
cat
pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading
Pseudomonas
strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.