bluuebunny/crossref_metadata_2025_split huggingface dataset:
Autocloning and Amplification of
LIP2
in
Yarrowia lipolytica
Authors: Pignède Georges , Wang Hui-Jie , Fudalej Franck , Seman Michel , Gaillardin Claude , Nicaud Jean-Marc 
Published: July 2002

ABSTRACT

We synthesized a
Yarrowia lipolytica
strain overproducing lipase for industrial applications by using long terminal repeat (ζ) of the
Y. lipolytica
retrotransposon Ylt1 and an allele of
URA3
with a promoter deletion to construct JMP3. JMP3 is a derivative of plasmid pHSS6 carrying a
Not
I-
Not
I cassette which contains a defective
URA3
allele, a polylinker sequence, and the ζ region for targeting to multiple sites in the genome of the recipient. We inserted the
LIP2
gene (encoding extracellular lipase) under the control of the strong
POX2
promoter into JMP3 to generate JMP6. The pHSS6 region was removed by
Not
I digestion prior to transformation. Two
Y. lipolytica
strains transformed with the JMP6
LIP2
cassette had a mean of 10 integrated copies devoid of the
Escherichia coli
region, corresponding to an autocloning event. The copy number in the transformants was stable even after 120 generations in nonselective and lipase-inducing conditions. The resulting strains could produce 0.5 g of active lipase per liter in the supernatant, 40 times more than the single-copy strain with the
LIP2
promoter. This work provides a new expression system in
Y. lipolytica
that results in strains devoid of bacterial DNA and in strains producing a high level of lipase for industrial uses, waste treatment, and pancreatic insufficiency therapy.
Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities
Authors: Dunbar John , Ticknor Lawrence O. , Kuske Cheryl R. 
Published: July 2002

ABSTRACT

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the
Proteobacteria
or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.
Development and Optimization of a Novel Immunomagnetic Separation- Bacteriophage Assay for Detection of
Salmonella enterica
Serovar Enteritidis in Broth
Authors: Favrin Stacy J. , Jassim Sabah A. , Griffiths Mansel W. 
Published: July 2002

ABSTRACT

Salmonella
is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of
Salmonella
in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of
Salmonella enterica
serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10
4
CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of
Salmonella
serovar Enteritidis in broth cultures which can be applied to preenriched food samples.
Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability
Authors: Ranjard L. , Poly F. , Lata J.-C. , Mougel C. , Thioulouse J. , Nazaret S. 
Published: July 2002

ABSTRACT
Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.
Visualization of
N
-Acylhomoserine Lactone-Mediated Cell-Cell Communication between Bacteria Colonizing the Tomato Rhizosphere
Authors: Steidle Anette , Sigl Katja , Schuhegger Regina , Ihring Alexandra , Schmid Markus , Gantner Stephan , Stoffels Marion , Riedel Kathrin , Givskov Michael , Hartmann Anton , Langebartels Christian , Eberl Leo 
Published: July 2002

ABSTRACT

Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing
N
-acyl-
l
-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of
Pseudomonas
putida
IsoF and
Serratia
liquefaciens
MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negative
P
.
putida
strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.
Analysis of the Type IV Fimbrial-Subunit Gene
fimA
of
Xanthomonas hyacinthi
: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths
Authors: van Doorn J. , Hollinger T. C. , Oudega B. 
Published: July 2002

ABSTRACT

A sensitive and specific detection method was developed for
Xanthomonas hyacinthi
; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene
fimA
from strain S148. The
fimA
gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA. The nucleotide sequence of
fimA
was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as
Xanthomonas campestris
pv. vesicatoria,
Neisseria gonorrhoeae
, and
Moraxella bovis
. In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of
fimA
, amplified a 226-bp DNA fragment in all
X. hyacinthi
isolates. This PCR was shown to be pathovar specific, as assessed by testing 71
Xanthomonas
pathovars and bacterial isolates belonging to other genera, such as
Erwinia
and
Pseudomonas
. Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in
X. hyacinthi
. Only two
Xanthomonas translucens
pathovars cross-reacted weakly in PCR. Primers amplifying a subsequence of the
fimA
gene of
X. campestris
pv. vesicatoria (T. Ojanen-Reuhs, N. Kalkkinen, B. Westerlund-Wikström, J. van Doorn, K. Haahtela, E.-L. Nurmiaho-Lassila, K. Wengelink, U. Bonas, and T. K. Korhonen, J. Bacteriol. 179: 1280–1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes. Under laboratory conditions, approximately 1,000 CFU of
X. hyacinthi
per ml could be detected. In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml. The results indicated that infected hyacinths with early symptoms could be successfully screened for
X. hyacinthi
with PCR.
Characterization of
Listeria monocytogenes
Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fingerprinting Techniques
Authors: Franciosa Giovanna , Tartaro Stefania , Wedell-Neergaard Christina , Aureli Paolo 
Published: July 2002

ABSTRACT

A total of 32
Listeria monocytogenes
strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated
L. monocytogenes
food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.
Natural Transformation of
Pseudomonas fluorescens
and
Agrobacterium tumefaciens
in Soil
Authors: Demanèche Sandrine , Kay Elisabeth , Gourbière François , Simonet Pascal 
Published: July 2002

ABSTRACT

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria,
Agrobacterium tumefaciens
and
Pseudomonas fluorescens
, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover,
P. fluorescens
produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested.
A. tumefaciens
could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.
The Leucine Incorporation Method Estimates Bacterial Growth Equally Well in Both Oxic and Anoxic Lake Waters
Authors: Bastviken David , Tranvik Lars 
Published: July 2002

ABSTRACT

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O
2
contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O
2
contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O
2
contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O
2
-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.
Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in
Acinetobacter
sp. Strain BD413 and
Thermus thermophilus
HB27
Authors: Friedrich Alexandra , Hartsch Thomas , Averhoff Beate 
Published: July 2002

ABSTRACT

The mesophile
Acinetobacter
sp. strain BD413 and the extreme thermophile
Thermus thermophilus
HB27 display high frequencies of natural transformation. In this study we identified and characterized a novel competence gene in
Acinetobacter
sp. strain BD413,
comA
, whose product displays significant similarities to the competence proteins ComA and ComEC in
Neisseria
and
Bacillus
species. Transcription of
comA
correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery. This finding strongly suggests that
comA
is part of a competence regulon. Examination of the genome sequence of
T. thermophilus
HB27 led to detection of a
comA/comEC
-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the
Bacillus subtilis
competence protein ComEA. To examine whether these two ORFs, designated
comEC
and
comEA
, are implicated in natural transformation of
T. thermophilus
HB27, both were disrupted by using a thermostable kanamycin resistance marker. Natural transformation in
comEC
mutants was reduced 1,000-fold, whereas in
comEA
mutants the natural transformation phenotype was completely eliminated. These results strongly suggest that both genes,
comEC
and
comEA
, are required for natural transformation in
T. thermophilus
HB27. Several transmembrane α-helices are predicted based on the amino acid sequences of ComA in
Acinetobacter
sp. strain BD413 and ComEC in
T. thermophilus
HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane.
Effects of Iron Limitation on the Degradation of Toluene by
Pseudomonas
Strains Carrying the TOL (pWWO) Plasmid
Authors: Dinkla Inez J. T. , Gabor Esther M. , Janssen Dick B. 
Published: July 2002

ABSTRACT

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by
Pseudomonas putida
mt2 and the transconjugant rhizosphere bacterium
P. putida
WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three
meta
-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer
o
-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.
Efficient System for Directed Integration into the
Lactobacillus acidophilus
and
Lactobacillus gasseri
Chromosomes via Homologous Recombination
Authors: Russell W. M. , Klaenhammer T. R. 
Published: July 2002

ABSTRACT

An efficient method is described for the generation of site-specific chromosomal integrations in
Lactobacillus acidophilus
and
Lactobacillus gasseri
. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35–50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the
Lactobacillus acidophilus
NCFM
lacL
gene encoding β-galactosidase and of the
Lactobacillus gasseri
ADH
gusA
gene encoding β-glucuronidase.
Molecular Characterization of a Thermostable Cyanophycin Synthetase from the Thermophilic Cyanobacterium
Synechococcus
sp. Strain MA19 and In Vitro Synthesis of Cyanophycin and Related Polyamides
Authors: Hai Tran , Oppermann-Sanio Fred Bernd , Steinbüchel Alexander 
Published: July 2002

ABSTRACT

The thermophilic cyanobacterium
Synechococcus
sp. strain MA19 contained the structural genes for cyanophycin synthetase (
cphA
) and cyanophycinase (
cphB
), which were identified, cloned, and sequenced in this study. The translation products of
cphA
and
cphB
exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as
Anabaena variabilis
and
Synechocystis
sp. Recombinant cells of
Escherichia coli
harboring
cphA
colinear with
lacPO
accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-β-
d
-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates,
l
-arginine and
l
-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, α-arginyl aspartic acid dipeptide, and poly-α,β-
dl
-aspartic acid as primers and also incorporated β-hydroxyaspartic acid instead of
l
-aspartic acid or
l
-canavanine instead of
l
-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.
Bacteriocin-Like Activity of
Butyrivibrio fibrisolvens
JL5 and Its Effect on Other Ruminal Bacteria and Ammonia Production
Authors: Rychlik Jennifer L. , Russell James B. 
Published: July 2002

ABSTRACT

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10
−6
. The 10
−8
and 10
−9
dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited
Clostridium sticklandii
SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to
Butyrivibrio fibrisolvens
B835. Strain JL5 inhibited
B. fibrisolvens
49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of
B. fibrisolvens
49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of
C. sticklandii
SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and
C. sticklandii
SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.
Widespread Distribution in Polar Oceans of a 16S rRNA Gene Sequence with Affinity to
Nitrosospira
-Like Ammonia-Oxidizing Bacteria
Authors: Hollibaugh James T. , Bano Nasreen , Ducklow Hugh W. 
Published: July 2002

ABSTRACT

We analyzed the phylogenetic compositions of ammonia-oxidizing bacteria of the β subclass of
Proteobacteria
from 42 Southern Ocean samples. We found a
Nitrosospira
-like 16S rRNA gene sequence in all 20 samples that yielded PCR products (8 of 30 samples from the Ross Sea and 12 of 12 samples from the Palmer Peninsula). We also found this sequence in Arctic Ocean samples, indicating a transpolar, if not global, distribution; however, slight differences between Arctic and Antarctic sequences may be evidence of polar endemism.