bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Resistance-Nodulation-Cell Division Transporter System Associated with the
syr-syp
Genomic Island of
Pseudomonas syringae
pv. syringae
Authors: Kang Hyojeung , Gross Dennis C. 
Published: September 2005

ABSTRACT

A tripartite resistance-nodulation-cell division (RND) transporter system, called the PseABC efflux system, was identified at the left border of the
syr
-
syp
genomic island of
Pseudomonas syringae
pv. syringae strain B301D. The PseABC efflux system was located within a 5.7-kb operon that encodes an outer membrane protein (PseA), a periplasmic membrane fusion protein (PseB), and an RND-type cytoplasmic membrane protein (PseC). The PseABC efflux system exhibited amino acid homology to a putative RND efflux system of
Ralstonia solanacearum
, with identities of 48% for PseA, 51% for PseB, and 61% for PseC. A nonpolar mutation within the
pseC
gene was generated by
nptII
insertional mutagenesis. The resultant mutant strain showed a larger reduction in syringopeptin secretion (67%) than in syringomycin secretion (41%) compared to parental strain B301D (
P
< 0.05). A β-glucuronidase assay with a
pseA
::
uidA
reporter construct indicated that the GacS/GacA two-component system controls expression of the
pseA
gene. Quantitative real-time reverse transcription-PCR was used to determine transcript levels of the syringomycin (
syrB1
) and syringopeptin (
sypA
) synthetase genes in strain B301D-HK4 (a
pseC
mutant). The expression of the
sypA
gene by mutant strain B301D-HK4 corresponded to approximately 13% of that by parental strain B301D, whereas the
syrB1
gene expression by mutant strain B301D-HK4 was nearly 61% (
P
< 0.05). In addition, the virulence of mutant strain B301D-HK4 for immature cherry fruits was reduced by about 58% compared to parental strain B301D (
P
< 0.05). Although the resistance of mutant strain B301D-HK4 to any antibiotic used in this study was not reduced compared to parental strain B301D, a drug-supersensitive
acrB
mutant of
Escherichia coli
showed two- to fourfold-increased resistance to acriflavine, erythromycin, and tetracycline upon heterologous expression of the
pseA
,
pseB
, and
pseC
genes (
pseABC
efflux genes). The PseABC efflux system is the first RND transporter system described for
P. syringae
, and it has an important role in secretion of syringomycin and syringopeptin.
Gene Cluster Responsible for Validamycin Biosynthesis in
Streptomyces hygroscopicus
subsp.
jinggangensis
5008
Authors: Yu Yi , Bai Linquan , Minagawa Kazuyuki , Jian Xiaohong , Li Lei , Li Jialiang , Chen Shuangya , Cao Erhu , Mahmud Taifo , Floss Heinz G. , Zhou Xiufen , Deng Zixin 
Published: September 2005

ABSTRACT

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from
Streptomyces hygroscopicus
subsp.
jinggangensis
5008 using heterologous probe
acbC
, a gene involved in the cyclization of
d
-sedoheptulose 7-phosphate to 2-
epi
-5-
epi
-valiolone of the acarbose biosynthetic gene cluster originated from
Actinoplanes
sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained
valA
(an
acbC
homologue encoding a putative cyclase) as well as two additional complete open reading frames (
valB
and
valC
, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of
d
-sedoheptulose 7-phosphate to 2-
epi
-5-
epi
-valiolone in vitro using the ValA protein heterologously overexpressed in
E. coli
. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.
Establishment of Tn
5096
-Based Transposon Mutagenesis in
Gordonia polyisoprenivorans
Authors: Banh Quyen , Arenskötter Matthias , Steinbüchel Alexander 
Published: September 2005

ABSTRACT

The transposons Tn
5
, Tn
10
, Tn
611
, and Tn
5096
were characterized regarding transposition in
Gordonia polyisoprenivorans
strain VH2. No insertional mutants were obtained employing Tn
5
or Tn
10
. The thermosensitive plasmid pCG79 harboring Tn
611
integrated into the chromosome of
G. polyisoprenivorans
; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn
5096
-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn
5096
as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(
cis
-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(
cis
-1,4-isoprene). One rubber-negative mutant was disrupted in
mcr
, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(
cis
-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.
Isolation of a Hop-Sensitive Variant ofLactobacillus lindneriand Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria
Authors: Suzuki Koji , Iijima Kazumaru , Ozaki Kazutaka , Yamashita Hiroshi 
Published: September 2005

ABSTRACTWe have isolated a hop-sensitive variant of the beer spoilage bacteriumLactobacillus lindneriDSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designatedhorBLandhorCLthat encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs ofhorBandhorC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those ofhorAand ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use ofhorB/horCandhorAis able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison ofhorBandhorChomologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution ofhorBandhorChomologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.
1
H Nuclear Magnetic Resonance Spectroscopy-Based Studies of the Metabolism of Food-Borne Carcinogen 2-Amino-3-Methylimidazo[4,5-
f
]Quinoline by Human Intestinal Microbiota
Authors: Humblot Christèle , Combourieu Bruno , Väisänen Marja-Liisa , Furet Jean-Pierre , Delort Anne-Marie , Rabot Sylvie 
Published: September 2005

ABSTRACT

2-Amino-3-methylimidazo[4,5-
f
]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 μM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ
1
H nuclear magnetic resonance (
1
H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and
1
H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction:
Bacteroides thetaiotaomicron
(
n
= 2),
Clostridium clostridiiforme
(
n
= 3),
Clostridium perfringens
(
n
= 1), and
Escherichia coli
(
n
= 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.
Improvement of the Fatty Acid Composition of an Oil-Producing Filamentous Fungus,
Mortierella alpina
1S-4, through RNA Interference with Δ12-Desaturase Gene Expression
Authors: Takeno Seiki , Sakuradani Eiji , Tomi Akiko , Inohara-Ochiai Misa , Kawashima Hiroshi , Ashikari Toshihiko , Shimizu Sakayu 
Published: September 2005

ABSTRACT

An oleaginous fungus,
Mortierella alpina
1S-4, is used commercially for arachidonic acid production. Δ12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in
M. alpina
1S-4, we silenced the Δ12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Δ12-desaturation-defective mutants previously identified. Thus, RNAi occurs in
M. alpina
and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.
New Chitosan-Degrading Strains That Produce Chitosanases Similar to ChoA of
Mitsuaria chitosanitabida
Authors: Yun ChoongSoo , Amakata Daiki , Matsuo Yasuhiro , Matsuda Hideyuki , Kawamukai Makoto 
Published: September 2005

ABSTRACT

The betaproteobacterium
Mitsuaria chitosanitabida
(formerly
Matsuebacter chitosanotabidus
) 3001 produces a chitosanase (ChoA) that is classified in glycosyl hydrolase family 80. While many chitosanase genes have been isolated from various bacteria to date, they show limited homology to the
M. chitosanitabida
3001 chitosanase gene (
choA
). To investigate the phylogenetic distribution of chitosanases analogous to ChoA in nature, we identified 67 chitosan-degrading strains by screening and investigated their physiological and biological characteristics. We then searched for similarities to ChoA by Western blotting and Southern hybridization and selected 11 strains whose chitosanases showed the most similarity to ChoA. PCR amplification and sequencing of the chitosanase genes from these strains revealed high deduced amino acid sequence similarities to ChoA ranging from 77% to 99%. Analysis of the 16S rRNA gene sequences of the 11 selected strains indicated that they are widely distributed in the β and γ subclasses of
Proteobacteria
and the
Flavobacterium
group. These observations suggest that the ChoA-like chitosanases that belong to family 80 occur widely in a broad variety of bacteria.
Colonization of Cattle Intestines byCampylobacter jejuniandCampylobacter lanienae
Authors: Inglis G. Douglas , Kalischuk Lisa D. , Busz Hilma W. , Kastelic John P. 
Published: September 2005

ABSTRACTThe location and abundance ofCampylobacter jejuniandCampylobacter lanienaein the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shedC. jejuniandC. lanienae(steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed.CampylobacterDNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. AlthoughC. jejuniandC. lanienaeDNA were detected in both animals, only steer A contained substantial quantities ofC. jejuniDNA. In both digesta and tissues of steer A,C. jejuniwas present in the duodenum and jejunum. Considerable quantities ofC. jejuniDNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities ofC. lanienaeDNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associatedC. lanienaeDNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance ofC. jejuniandC. lanienaeDNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA ofC. jejuniandC. lanienaewere detected in the digesta of 57% and 95% of the animals, respectively.C. jejuniassociated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance ofC. jejuniDNA in the ileum and rectum.C. lanienaewas most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore,C. jejunicolonized the proximal small intestine of asymptomatic beef cattle, whereasC. lanienaeprimarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.
Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression
Authors: He Zhili , Wu Liyou , Fields Matthew W. , Zhou Jizhong 
Published: September 2005

ABSTRACT

Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a whole-genome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from
Shewanella oneidensis
MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. Hybridizations conducted at 42°C, 45°C, 50°C, and 60°C indicated that good sensitivities were obtained at 45°C for oligonucleotide probes in the presence of 50% formamide, under which conditions specific signals were detected by both PCR and oligonucleotide probes. Signal intensity increased as the length of the oligonucleotide probe increased, and the 70-mer oligonucleotide probes produced signal intensities similar to the intensities obtained with the PCR probes and detected numbers of open reading frames similar to the numbers detected with the PCR probes. PCR amplicon, 70-mer, 60-mer, and 50-mer arrays had detection sensitivities of 5.0, 25, 100, and 100 ng of genomic DNA, which were equivalent to approximately 1.9 × 10
6
, 9.2 × 10
6
, 3.7 × 10
7
, and 3.7 × 10
7
copies, respectively, when the array was hybridized with genomic DNA. To evaluate differential gene expression under experimental conditions,
S. oneidensis
MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.
Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in
Staphylococcus haemolyticus
Grown in Toluene
Authors: Nielsen Lindsey E. , Kadavy Dana R. , Rajagopal Soumitra , Drijber Rhae , Nickerson Kenneth W. 
Published: September 2005

ABSTRACT

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of
Staphylococcus haemolyticus
which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and
p
-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus,
S. haemolyticus
alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in
S. haemolyticus
is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of
Staphylococcus aureus
and five strains of
Staphylococcus epidermidis
, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.
The Microcystin Composition of the Cyanobacterium
Planktothrix agardhii
Changes toward a More Toxic Variant with Increasing Light Intensity
Authors: Tonk Linda , Visser Petra M. , Christiansen Guntram , Dittmann Elke , Snelder Eveline O. F. M. , Wiedner Claudia , Mur Luuc R. , Huisman Jef 
Published: September 2005

ABSTRACT

The cyanobacterium
Planktothrix agardhii
, which is dominant in many shallow eutrophic lakes, can produce hepatotoxic microcystins. Currently, more than 70 different microcystin variants have been described, which differ in toxicity. In this study, the effect of photon irradiance on the production of different microcystin variants by
P. agardhii
was investigated using light-limited turbidostats. Both the amount of the mRNA transcript of the
mcyA
gene and the total microcystin production rate increased with photon irradiance up to 60 μmol m
−2
s
−1
, but they started to decrease with irradiance greater than 100 μmol m
−2
s
−1
. The cellular content of total microcystin remained constant, independent of the irradiance. However, of the two main microcystin variants detected in
P. agardhii
, the microcystin-DeRR content decreased twofold with increased photon irradiance, whereas the microcystin-DeLR content increased threefold. Since microcystin-DeLR is considerably more toxic than microcystin-DeRR, this implies that
P. agardhii
becomes more toxic at high light intensities.
13
C-Carrier DNA Shortens the Incubation Time Needed To Detect Benzoate-Utilizing Denitrifying Bacteria by Stable-Isotope Probing
Authors: Gallagher E. , McGuinness L. , Phelps C. , Young L. Y. , Kerkhof L. J. 
Published: September 2005

ABSTRACT

The active bacterial community able to utilize benzoate under denitrifying conditions was elucidated in two coastal sediments using stable-isotope probing (SIP) and
nosZ
gene amplification. The SIP method employed samples from Norfolk Harbor, Virginia, and a Long-Term Ecosystem Observatory (no. 15) off the coast of Tuckerton, New Jersey. The SIP method was modified by use of archaeal carrier DNA in the density gradient separation. The carrier DNA significantly reduced the incubation time necessary to detect the
13
C-labeled bacterial DNA from weeks to hours in the coastal enrichments. No denitrifier DNA was found to contaminate the archaeal
13
C-carrier when [
12
C]benzoate was used as a substrate in the sediment enrichments. Shifts in the activity of the benzoate-utilizing denitrifying population could be detected throughout a 21-day incubation. These results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader(s) in a bacterial population and to document the cross-feeding microbial community.
Quorum-Sensing Mutations Affect Attachment and Stability of
Burkholderia cenocepacia
Biofilms
Authors: Tomlin Kerry L. , Malott Rebecca J. , Ramage Gordon , Storey Douglas G. , Sokol Pamela A. , Ceri H. 
Published: September 2005

ABSTRACT

Biofilm formation in
Burkholderia cenocepacia
has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the
cepIR
and
cciIR
quorum-sensing systems of
Burkholderia cenocepacia
K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the
cepI
and
cepR
mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the
cciI
mutant nor the double
cciI cepI
mutant was deficient in attachment, and restoration of the
cciI
gene resulted in less attachment than for the mutants. Meanwhile, the
cciR
mutant did show a significant reduction in attachment, as did the
cciR cepIR
mutant. While there was no change in antibiotic susceptibility with the individual
cepIR
and
cciIR
mutants, the
cepI cciI
mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the
cepI
and
cepR
mutants. Flow cell analysis of the individual
cepIR
mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of
B. cenocepacia
that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.