bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dual Role of Response Regulator StyR in Styrene Catabolism Regulation
Authors: Leoni Livia , Rampioni Giordano , Di Stefano Valeria , Zennaro Elisabetta 
Published: September 2005

ABSTRACT

In
Pseudomonas fluorescens
ST, the promoter of the styrene catabolic operon, P
styA
, is induced by styrene and repressed by the addition of preferred carbon sources. P
styA
is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in P
styA
regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on P
styA
. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the P
styA
promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of P
styA
. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions.
Genetic Determinants of Volatile-Thiol Release bySaccharomyces cerevisiaeduring Wine Fermentation
Authors: Howell Kate S. , Klein Mathias , Swiegers Jan H. , Hayasaka Yoji , Elsey Gordon M. , Fleet Graham H. , Høj Peter B. , Pretorius Isak S. , de Barros Lopes Miguel A. 
Published: September 2005

ABSTRACTVolatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation,Saccharomyces cerevisiaemediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.
Hydroxylation and Carboxylation—Two Crucial Steps of Anaerobic Benzene Degradation by
Dechloromonas
Strain RCB
Authors: Chakraborty Romy , Coates John D. 
Published: September 2005

ABSTRACT

Benzene is a highly toxic industrial compound that is essential to the production of various chemicals, drugs, and fuel oils. Due to its toxicity and carcinogenicity, much recent attention has been focused on benzene biodegradation, especially in the absence of molecular oxygen. However, the mechanism by which anaerobic benzene biodegradation occurs is still unclear. This is because until the recent isolation of
Dechloromonas
strains JJ and RCB no organism that anaerobically degraded benzene was available with which to elucidate the pathway. Although many microorganisms use an initial fumarate addition reaction for hydrocarbon biodegradation, the large activation energy required argues against this mechanism for benzene. Other possible mechanisms include hydroxylation, carboxylation, biomethylation, or reduction of the benzene ring, but previous studies performed with undefined benzene-degrading cultures were unable to clearly distinguish which, if any, of these alternatives is used. Here we demonstrate that anaerobic nitrate-dependent benzene degradation by
Dechloromonas
strain RCB involves an initial hydroxylation, subsequent carboxylation, and loss of the hydroxyl group to form benzoate. These studies provide the first pure-culture evidence of the pathway of anaerobic benzene degradation. The outcome of these studies also suggests that all anaerobic benzene-degrading microorganisms, regardless of their terminal electron acceptor, may use this pathway.
Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase
Authors: Yanase Michiyo , Takata Hiroki , Fujii Kazutoshi , Takaha Takeshi , Kuriki Takashi 
Published: September 2005

ABSTRACT
The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.
Novel Chemoautotrophic Endosymbiosis between a Member of the
Epsilonproteobacteria
and the Hydrothermal-Vent Gastropod
Alviniconcha
aff.
hessleri
(Gastropoda: Provannidae) from the Indian Ocean
Authors: Suzuki Yohey , Sasaki Takenori , Suzuki Masae , Nogi Yuichi , Miwa Tetsuya , Takai Ken , Nealson Kenneth H. , Horikoshi Koki 
Published: September 2005

ABSTRACT

The hydrothermal-vent gastropod
Alviniconcha
aff.
hessleri
from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the
Proteobacteria
. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all
13
C enriched relative to the gastropod biomass and that the monounsaturated C
16
fatty acid that originates from the endosymbiont is as
13
C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from
13
C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for
Alviniconcha
gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.
Design and Evaluation of 16S rRNA-Targeted Peptide Nucleic Acid Probes for Whole-Cell Detection of Members of the Genus
Listeria
Authors: Brehm-Stecher Byron F. , Hyldig-Nielsen Jens J. , Johnson Eric A. 
Published: September 2005

ABSTRACT

Six fluorescein-labeled peptide nucleic acid oligomers targeting
Listeria
-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22
Listeria
strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both
Listeria
spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml
−1
. LisUn-11 was the brightest probe and stained all six
Listeria
species. LisUn-3 hybridized with all
Listeria
spp. except for
L. grayi
, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with
Listeria
spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of
Listeria
spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control
L. monocytogenes
.
Interaction of
Pasteurella multocida
with Free-Living Amoebae
Authors: Hundt Matthew J. , Ruffolo Carmel G. 
Published: September 2005

ABSTRACT

Pasteurella multocida
is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between
P. multocida
and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing
P. multocida
strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of
P. multocida
within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between
P. multocida
and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between
P. multocida
and host cells. Free-living amoebae are ubiquitous within water and soil environments, and
P. multocida
has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between
P. multocida
and amoebae may occur within the natural environment.
Generation and Phenotypic Characterization of
Aspergillus nidulans
Methylisocitrate Lyase Deletion Mutants: Methylisocitrate Inhibits Growth and Conidiation
Authors: Brock Matthias 
Published: September 2005

ABSTRACT

Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of
Aspergillus nidulans
was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the α-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2
R
,3
S
)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the

K
i

of (2
R
,3
S
)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 μM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.
Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments
Authors: McClay Kevin , Boss Corinne , Keresztes Ivan , Steffan Robert J. 
Published: September 2005

ABSTRACT

Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of
tmoA
, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of
Burkholderia cepacia
G4. These results demonstrate that modification of the
tmoA
active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.
Spatial Organization of Dual-Species Bacterial Aggregates on Leaf Surfaces
Authors: Monier J.-M. , Lindow S. E. 
Published: September 2005

ABSTRACT

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves,
Pseudomonas syringae
,
Pantoea agglomerans
, and
Pseudomonas fluorescens
. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of
P. fluorescens
A506 and
P. agglomerans
299R and mixtures of
P. syringae
B728a and
P. agglomerans
299R than in mixtures of two isogenic strains of
P. agglomerans
299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a
gfp
-marked strain of
P. agglomerans
299R and a
cfp
-marked strain of
P. agglomerans
299R, or of
P. fluorescens
A506 and
P. agglomerans
299R, were similar. However, the proportion of dead cells in mixed aggregates of
P. syringae
B728a and
P. agglomerans
299R was significantly higher (13.2% ± 8.2%) than that in monospecific aggregates of these two strains (1.6% ± 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the
P. agglomerans
299R cells located at the interface were dead, while only about 24% of the
P. syringae
B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.
Whole-Genome Shotgun Optical Mapping of
Rhodospirillum rubrum
Authors: Reslewic Susan , Zhou Shiguo , Place Mike , Zhang Yaoping , Briska Adam , Goldstein Steve , Churas Chris , Runnheim Rod , Forrest Dan , Lim Alex , Lapidus Alla , Han Cliff S. , Roberts Gary P. , Schwartz David C. 
Published: September 2005

ABSTRACT

Rhodospirillum rubrum
is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of
R. rubrum
strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.
rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria
Authors: Kenzaka Takehiko , Ishidoshiro Ai , Yamaguchi Nobuyasu , Tani Katsuji , Nasu Masao 
Published: September 2005

ABSTRACT

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain
Bacteria
were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of
Cytophaga-Flavobacterium
existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.
Complexation of Uranium by Cells and S-Layer Sheets of
Bacillus sphaericus
JG-A12
Authors: Merroun Mohamed L. , Raff Johannes , Rossberg André , Hennig Christoph , Reich Tobias , Selenska-Pobell Sonja 
Published: September 2005

ABSTRACT

Bacillus sphaericus
JG-A12 is a natural isolate recovered from a uranium mining waste pile near the town of Johanngeorgenstadt in Saxony, Germany. The cells of this strain are enveloped by a highly ordered crystalline proteinaceous surface layer (S-layer) possessing an ability to bind uranium and other heavy metals. Purified and recrystallized S-layer proteins were shown to be phosphorylated by phosphoprotein-specific staining, inductive coupled plasma mass spectrometry analysis, and a colorimetric method. We used extended X-ray absorption fine-structure (EXAFS) spectroscopy to determine the structural parameters of the uranium complexes formed by purified and recrystallized S-layer sheets of
B. sphaericus
JG-A12. In addition, we investigated the complexation of uranium by the vegetative bacterial cells. The EXAFS analysis demonstrated that in all samples studied, the U(VI) is coordinated to carboxyl groups in a bidentate fashion with an average distance between the U atom and the C atom of 2.88 ± 0.02 Å and to phosphate groups in a monodentate fashion with an average distance between the U atom and the P atom of 3.62 ± 0.02 Å. Transmission electron microscopy showed that the uranium accumulated by the cells of this strain is located in dense deposits at the cell surface.