bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host
Authors: Young Eric , Poucher Ashley , Comer Austin , Bailey Alexandra , Alper Hal 
Published: March 2011

ABSTRACT

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in
Saccharomyces cerevisiae
. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant
Saccharomyces cerevisiae
host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of
Scheffersomyces stipitis
and open reading frames from the yeasts
Yarrowia lipolytica
and
Debaryomyces hansenii.
Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of
D. hansenii
XylHP
and
S. stipitis
XUT1
and
XUT3
demonstrated moderate transport efficiency and higher xylose preferences.
Substrate Specificity of the Bacillus licheniformis Lyxose Isomerase YdaE and Its Application in
In Vitro
Catalysis for Bioproduction of Lyxose and Glucose by Two-Step Isomerization
Authors: Patel Darshan H. , Wi Seung Gon , Lee Seong-Gene , Lee Dae-Seok , Song Youn-ho , Bae Hyeun-Jong 
Published: March 2011

ABSTRACT

Enzymatic processes are useful for industrially important sugar production, and
in vitro
two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by
ydaE
of
Bacillus licheniformis
was found to have broad substrate specificities and has shown high catalytic efficiency on
d
-lyxose, suggesting that the enzyme is
d
-lyxose isomerase.
Escherichia coli
BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn
2+
. The apparent

K
m

values for
d
-lyxose and
d
-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (
k
cat
/

K
m

) for lyxose (3.2 ± 0.1 mM
−1
s
−1
) was higher than that for
d
-mannose (1.6 mM
−1
s
−1
). The purified protein was applied to the bioproduction of
d
-lyxose and
d
-glucose from
d
-xylose and
d
-mannose, respectively, along with the thermostable xylose isomerase of
Thermus thermophilus
HB08. From an initial concentration of 10 mM
d
-lyxose and
d
-mannose, 3.7 mM and 3.8 mM
d
-lyxose and
d
-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for
in vitro
catalysis and can be applied to industrial production.
Assessing and Improving Methods Used in Operational Taxonomic Unit-Based Approaches for 16S rRNA Gene Sequence Analysis
Authors: Schloss Patrick D. , Westcott Sarah L. 
Published: March 2011

ABSTRACT
In spite of technical advances that have provided increases in orders of magnitude in sequencing coverage, microbial ecologists still grapple with how to interpret the genetic diversity represented by the 16S rRNA gene. Two widely used approaches put sequences into bins based on either their similarity to reference sequences (i.e., phylotyping) or their similarity to other sequences in the community (i.e., operational taxonomic units [OTUs]). In the present study, we investigate three issues related to the interpretation and implementation of OTU-based methods. First, we confirm the conventional wisdom that it is impossible to create an accurate distance-based threshold for defining taxonomic levels and instead advocate for a consensus-based method of classifying OTUs. Second, using a taxonomic-independent approach, we show that the average neighbor clustering algorithm produces more robust OTUs than other hierarchical and heuristic clustering algorithms. Third, we demonstrate several steps to reduce the computational burden of forming OTUs without sacrificing the robustness of the OTU assignment. Finally, by blending these solutions, we propose a new heuristic that has a minimal effect on the robustness of OTUs and significantly reduces the necessary time and memory requirements. The ability to quickly and accurately assign sequences to OTUs and then obtain taxonomic information for those OTUs will greatly improve OTU-based analyses and overcome many of the challenges encountered with phylotype-based methods.
Genetic Screening Strategy for Rapid Access to Polyether Ionophore Producers and Products in Actinomycetes
Authors: Wang Hao , Liu Ning , Xi Lijun , Rong Xiaoying , Ruan Jisheng , Huang Ying 
Published: March 2011

ABSTRACT

Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of
Actinomycetales
. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including
Micromonospora
, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.
Comparative Genomics of Escherichia coli Strains Causing Urinary Tract Infections
Authors: Vejborg Rebecca Munk , Hancock Viktoria , Schembri Mark A. , Klemm Per 
Published: March 2011

ABSTRACT

The virulence determinants of uropathogenic
Escherichia coli
have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range of different clinical backgrounds, i.e., urosepsis, pyelonephritis, cystitis, and asymptomatic bacteriuria (ABU), using comparative genomic hybridization analysis. A microarray based on 31 complete
E. coli
sequences was used. It emerged that there is little correlation between the genotypes of the strains and their disease categories but strong correlation between the genotype and the phylogenetic group association. Also, very few genetic differences may exist between isolates causing symptomatic and asymptomatic infections. Only relatively few genes that could potentially differentiate between the individual disease categories were identified. Among these were two genomic islands, namely, pathogenicity island (PAI)-CFT073-
serU
and PAI-CFT073-
pheU
, which were significantly more associated with the pyelonephritis and urosepsis isolates than with the ABU and cystitis isolates. These two islands harbor genes encoding virulence factors, such as P fimbriae (pyelonephritis-associated fimbriae) and an important immunomodulatory protein, TcpC. It seems that both urovirulence and growth fitness can be attributed to an assortment of genes rather than to a specific gene set. Taken together, urovirulence and fitness are the results of the interplay of a mixture of factors taken from a rich menu of genes.
Characterization of Putative Virulence Genes on the Related RepFIB Plasmids Harbored by Cronobacter spp
Authors: Franco A. A. , Hu L. , Grim C. J. , Gopinath G. , Sathyamoorthy V. , Jarvis K. G. , Lee C. , Sadowski J. , Kim J. , Kothary M. H. , McCardell B. A. , Tall B. D. 
Published: March 2011

ABSTRACT

Cronobacter
spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus
Chronobacter
consists of six species:
C. sakazakii
,
C. malonaticus
,
C. muytjensii
,
C. turicensis
,
C. dublinensis
, and
Cronobacter
genomospecies group 1. Whole-genome sequencing of
C. sakazakii
BAA-894 and
C. turicensis
z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively.
In silico
analysis showed that both plasmids encode a single RepFIB-like origin of replication gene,
repA
, as well as two iron acquisition systems (
eitCBAD
and
iucABCD/iutA
). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a
cpa
(
Cronobacter
plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (
fhaB
), its specifc transporter gene (
fhaC
), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a
repA
-targeted PCR assay, 97% of 229
Cronobacter
species isolates were found to possess a homologous RepFIB plasmid. All
repA
PCR-positive strains were also positive for the
eitCBAD
and
iucABCD/iutA
iron acquisition systems. However, the presence of
cpa
, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.
Efficient Extension of Slipped DNA Intermediates by DinB Is Required To Escape Primer Template Realignment by DnaQ
Authors: Foti James J. , Walker Graham C. 
Published: March 2011

ABSTRACT

We show that
Escherichia coli
DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the −4 position. A DinB(Y79L) variant, which extends these substrates less efficiently
in vitro
, allows the proofreading function of polymerase III to reverse their formation
in vivo
.
The Y-Family DNA Polymerase Dpo4 Uses a Template Slippage Mechanism To Create Single-Base Deletions
Authors: Wu Yifeng , Wilson Ryan C. , Pata Janice D. 
Published: March 2011

ABSTRACT

The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5′ flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of
Sulfolobus
. To reconcile these apparent differences, we have characterized Dpo4 from
Sulfolobus solfataricus
using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from
Sulfolobus acidocaldarius
. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.
An Agrobacterium VirB10 Mutation Conferring a Type IV Secretion System Gating Defect
Authors: Banta Lois M. , Kerr Jennifer E. , Cascales Eric , Giuliano Meghan E. , Bailey Megan E. , McKay Cedar , Chandran Vidya , Waksman Gabriel , Christie Peter J. 
Published: March 2011

ABSTRACT

Agrobacterium
VirB7, VirB9, and VirB10 form a “core complex” during biogenesis of the VirB/VirD4 type IV secretion system (T4SS). VirB10 spans the cell envelope and, in response to sensing of ATP energy consumption by the VirB/D4 ATPases, undergoes a conformational change required for DNA transfer across the outer membrane (OM). Here, we tested a model in which VirB10 regulates substrate passage by screening for mutations that allow for unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact. One mutation, G272R, conferred VirE2 release and also rendered VirB10 conformationally insensitive to cellular ATP depletion. Strikingly, G272R did not affect substrate transfer to target cells (Tra
+
) but did block pilus production (Pil
−
). The G272R mutant strain displayed enhanced sensitivity to vancomycin and SDS but did not nonspecifically release periplasmic proteins or VirE2 truncated of its secretion signal. G272 is highly conserved among VirB10 homologs, including pKM101 TraF, and in the TraF X-ray structure the corresponding Gly residue is positioned near an α-helical domain termed the antenna projection (AP), which is implicated in formation of the OM pore. A partial AP deletion mutation (ΔAP) also confers a Tra
+
Pil
−
phenotype; however, this mutation did not allow VirE2 surface exposure but instead allowed the release of pilin monomers or short oligomers to the milieu. We propose that (i) G272R disrupts a gating mechanism in the core chamber that regulates substrate passage across the OM and (ii) the G272R and ΔAP mutations block pilus production at distinct steps of the pilus biogenesis pathway.
Inactivation of σ
F
in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes σ
E
and σ
G
Protein Expression but Does Not Block Solvent Formation
Authors: Jones Shawn W. , Tracy Bryan P. , Gaida Stefan M. , Papoutsakis Eleftherios T. 
Published: March 2011

ABSTRACT

Clostridium acetobutylicum
is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation—both stationary-phase events—remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the
sigF
gene in
C. acetobutylicum
was successfully disrupted and silenced. Not only σ
F
but also the sigma factors σ
E
and σ
G
were not detected in the
sigF
mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the
spoIIA
operon (
spoIIAA
-
spoIIAB
-
sigF
) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ
F
expression was restored along with sporulation and σ
E
and σ
G
protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (
csfB
,
gpr
,
spoIIP
,
sigG
,
lonB
, and
spoIIR
) that could be controlled by σ
F
, based on the
Bacillus subtilis
model, indicated that
sigG
may be under the control of σ
F
, but
spoIIR
, an important activator of σ
E
in
B. subtilis
, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome—here, the
C. acetobutylicum
chromosome—with the aim of altering cell metabolism and differentiation.
Development and Use of a Gene Deletion Strategy for Flavobacterium johnsoniae To Identify the Redundant Gliding Motility Genes
remF
,
remG
,
remH
, and
remI
Authors: Rhodes Ryan G. , Pucker Halley G. , McBride Mark J. 
Published: March 2011

ABSTRACT

Cells of
Flavobacterium johnsoniae
exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the
sprB
operon (
sprC
,
sprD
,
sprB
, and
sprF
) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (
remF
,
remG
, and
fjoh
_
0982
) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant
rpsL
mutant of
F. johnsoniae
was isolated. Introduction of wild-type
rpsL
on a plasmid restored streptomycin sensitivity, demonstrating that wild-type
rpsL
is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type
rpsL
and used streptomycin for counterselection. This approach was used to delete the region spanning
remF
,
remG
, and
fjoh
_
0982
. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of
remF
(
remH
) and a paralog of
remG
(
remI
). Deletion of
remH
and
remI
had no effect on motility of wild-type cells, but cells lacking
remF
and
remH
, or cells lacking
remG
and
remI
, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The
rpsL
counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of
F. johnsoniae
physiology.
Overexpression of
pknE
Blocks Heterocyst Development in Anabaena sp. Strain PCC 7120
Authors: Saha Sushanta K. , Golden James W. 
Published: March 2011

ABSTRACT

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334,
pknE
(alr3732), all4668, and all4838, were fused to a
gfpmut2
reporter gene to determine their expression during heterocyst development in the cyanobacterium
Anabaena
(
Nostoc
) sp. strain PCC 7120. P

pknE

-gfp
was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of
pknE
required a 118-bp upstream region and was abolished in a
hetR
mutant. A
pknE
mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of
pknE
from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of
pknE
from the copper-inducible
petE
promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing
pknE
and containing P

hetR

-gfp
or P

patS

-gfp
reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of
pknE
blocks HetR activity or downstream regulation.
Biosynthesis and Regulation of Grisemycin, a New Member of the Linaridin Family of Ribosomally Synthesized Peptides Produced by Streptomyces griseus IFO 13350
Authors: Claesen Jan , Bibb Mervyn J. 
Published: March 2011

ABSTRACT

Our recent identification and genetic analysis of the biosynthetic gene cluster for production of the ribosomally synthesized and posttranslationally modified peptide cypemycin revealed a new class of peptide natural products, the linaridins. Here we describe the identification and characterization of grisemycin, a linaridin produced by a previously unidentified gene cluster in
Streptomyces griseus
IFO 13350. Mass spectrometric analysis revealed that grisemycin possesses at least three of the modifications found in cypemycin, as well as an analogous leader peptidase cleavage site. Expression of putative grisemycin biosynthetic genes in a
Streptomyces coelicolor
A3(2) derivative, combined with deletion of the gene encoding the grisemycin precursor peptide, confirmed the identity of the grisemycin gene cluster. Both grisemycin and cypemycin depend on the transcriptional activator AdpA for wild-type levels of production.
Draft Genome Sequence of Caloramator australicus Strain RC3
T
, a Thermoanaerobe from the Great Artesian Basin of Australia
Authors: Ogg Christopher D. , Patel Bharat K. C. 
Published: March 2011

ABSTRACT

Caloramator australicus
strain RC3
T
(JCM 15081
T
= KCTC 5601
T
) is the type strain of a newly identified thermophilic species, which was isolated from red microbial mats that thrive at 66°C in the runoff channel of a Great Artesian Basin bore (New Lorne bore, registered number 17263) in outback Queensland, Australia. The ability of the
C. australicus
strain to use metals as terminal electron acceptors has led to concerns that it could colonize and enhance corrosion of the metal casing of Great Artesian Basin bore well pipes and that this could subsequently lead to bore failure and loss of water availability for the community which is so reliant on it. The genome of the
C. australicus
strain has been sequenced, and annotation of the ∼2.65-Mb sequence indicates that the attributes are consistent with physiological and phenotypic traits.