bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Genomic Species in
Agrobacterium
Biovar 1 by AFLP Genomic Markers
Authors: Portier Perrine , Fischer-Le Saux Marion , Mougel Christophe , Lerondelle Catherine , Chapulliot David , Thioulouse Jean , Nesme Xavier 
Published: November 2006

ABSTRACT

Biovar 1 of the genus
Agrobacterium
consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as
Agrobacterium
ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of
Agrobacterium tumefaciens
), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely “species-specific” markers tagging the core genomes of the species.
Culture Isolation and Culture-Independent Clone Libraries Reveal New MarineSynechococcusEcotypes with Distinctive Light and N Physiologies
Authors: Ahlgren Nathan A. , Rocap Gabrielle 
Published: November 2006

ABSTRACTMarine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacteriumSynechococcuscan be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of aSynechococcuscommunity from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of sevenSynechococcusphylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed amongSynechococcusstrains than previously known. Thus, as in its sister genusProchlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marineSynechococcuslineage.
Cell Surface Expression of Bacterial Esterase A by
Saccharomyces cerevisiae
and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response
Authors: Breinig Frank , Diehl Björn , Rau Sabrina , Zimmer Christian , Schwab Helmut , Schmitt Manfred J. 
Published: November 2006

ABSTRACT

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from
Burkholderia gladioli
was covalently anchored into the cell wall of
Saccharomyces cerevisiae
by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When
p
-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg
−1
protein for Kre1/EstA/Cwp2p and 72 mU mg
−1
protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg
−1
protein for Kre1/EstA/Cwp2p and 1.27 U mg
−1
protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in
Escherichia coli
, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.
Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community
▿
Authors: Singh Brajesh K. , Nazaries Loic , Munro Stacey , Anderson Ian C. , Campbell Colin D. 
Published: November 2006

ABSTRACT

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub
Calluna vulgaris
growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with
Vaccinium myrtillus
collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.
Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils
Authors: Oline David K. 
Published: November 2006

ABSTRACT
I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale (∼100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.
Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity
Authors: Ben-Dov Eitan , Shapiro Orr H. , Siboni Nachshon , Kushmaro Ariel 
Published: November 2006

ABSTRACTTo overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively toProteobacteria(95.8%). Sequences belonging toFirmicutes(42.6%) andThermotogae(9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, whileProteobacteriasequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.
An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments
Authors: Lloyd Karen G. , Lapham Laura , Teske Andreas 
Published: November 2006

ABSTRACT

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as
mcrA
gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 μM coexist with sulfate concentrations around 10 mM. Clone libraries of
dsrAB
genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.
Spatiometabolic Stratification of
Shewanella oneidensis
Biofilms
Authors: Teal Tracy K. , Lies Douglas P. , Wold Barbara J. , Newman Dianne K. 
Published: November 2006

ABSTRACT

Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that
Shewanella oneidensis
biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature
S. oneidensis
biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.
The Mosaic Nature of Intergenic 16S-23S rRNA Spacer Regions Suggests rRNA Operon Copy Number Variation in
Clostridium difficile
Strains
Authors: Sadeghifard Nourkhoda , Gürtler Volker , Beer Michael , Seviour Robert J. 
Published: November 2006

ABSTRACT

Clostridium difficile
is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of
C. difficile
to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In
C. difficile
strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8
rrn c
opies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA
Ala
gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the
rrn
operons in different strains and different
rrn
copies in a single strain but also among ISRs of the same length. It appears that
C. difficile
ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable
rrn
copies within and between five different strains of
C. difficile
.
Effects of Mutations in the Substrate-Binding Domain of Poly[(
R
)-3-Hydroxybutyrate] (PHB) Depolymerase from
Ralstonia pickettii
T1 on PHB Degradation
Authors: Hiraishi Tomohiro , Hirahara Yoko , Doi Yoshiharu , Maeda Mizuo , Taguchi Seiichi 
Published: November 2006

ABSTRACT

Poly[(
R
)-3-hydroxybutyrate] (PHB) depolymerase from
Ralstonia pickettii
T1 (PhaZ
RpiT1
) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ
RpiT1
was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ
RpiT1
. Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ
RpiT1
adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.
Engineering of Primary Carbohydrate Metabolism for Increased Production of Actinorhodin in
Streptomyces coelicolor
Authors: Ryu Yong-Gu , Butler Michael J. , Chater Keith F. , Lee Kye Joon 
Published: November 2006

ABSTRACT

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in
Streptomyces coelicolor
. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by
zwf2
plays a more important role than that encoded by
zwf1
in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The
pgm
-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in
S. coelicolor
.
Identification of Potentially Human-Pathogenic
Enterocytozoon bieneusi
Genotypes in Various Birds
Authors: Lobo Maria Luísa , Xiao Lihua , Cama Vitaliano , Magalhães Nuno , Antunes Francisco , Matos Olga 
Published: November 2006

ABSTRACT

Enterocytozoon bieneusi
was detected in 24 of 83 samples from birds of the orders Columbiformes, Passeriformes, and Psittaciformes. It was identical to or closely related to the Peru6 genotype, which was previously found in humans in Peru. Thus, various birds can be a significant source of environmental contamination by potentially human-pathogenic
E. bieneusi
.
Mechanism of Augmentation of Organotin Decomposition by Ferripyochelin: Formation of Hydroxyl Radical and Organotin-Pyochelin-Iron Ternary Complex
Authors: Sun Guo-Xin , Zhong Jian-Jiang 
Published: November 2006

ABSTRACT

Pyochelin (PCH), a kind of siderophore secreted by
Pseudomonas aeruginosa
, was recently found to have triphenyltin (TPT)-decomposing capacity. In this work, significant augmentation of TPT decomposition by ferripyochelin (FePCH), the chelating compound of PCH with iron, was demonstrated in Tris-HCl buffer (pH 8.0). The generation of hydroxyl radical (HO·) in the presence of FePCH was observed. Inhibition of HO· generation by adding catalase and HO· scavengers (methanol and dimethyl sulfoxide) decreased TPT decomposition, while an increase in HO· formation in the presence of H
2
O
2
enhanced its decomposition. Our findings indicated that HO· generated in the reaction system was responsible for the enhanced TPT decomposition by FePCH versus PCH. The existence of the TPT-pyochelin-iron ternary complex was demonstrated by electron spray ionization-mass spectrometry, tandem mass spectrometry, and
1
H nuclear magnetic resonance. On the basis of the above results, HO· produced in the presence of FePCH was deduced to be in close proximity to TPT and has more opportunity to attack the Sn-C bond, which resulted in the enhanced organotin decomposition. The information obtained may have considerable environmental significance.
Genetic Characterization of the Resorcinol Catabolic Pathway in
Corynebacterium glutamicum
Authors: Huang Yan , Zhao Ke-Xin , Shen Xi-Hui , Chaudhry Muhammad Tausif , Jiang Cheng-Ying , Liu Shuang-Jiang 
Published: November 2006

ABSTRACT

Corynebacterium glutamicum
grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in
Escherichia coli
revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in
E. coli
revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor.
E. coli
cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by
C. glutamicum
. The function of NCgl2953 remains unclear.