bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two-Plasmid Vector System for Independently Controlled Expression of Green and Red Fluorescent Fusion Proteins in Staphylococcus aureus
Authors: Brzoska Anthony J. , Firth Neville 
Published: March 2013

ABSTRACT

We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in
Staphylococcus aureus
. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.
Characterization of a Regulatory Network of Peptide Antibiotic Detoxification Modules in Lactobacillus casei BL23
Authors: Revilla-Guarinos Ainhoa , Gebhard Susanne , Alcántara Cristina , Staroń Anna , Mascher Thorsten , Zúñiga Manuel 
Published: March 2013

ABSTRACT

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E).
Lactobacillus casei
BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to
L. casei
resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the
dlt
operon (
d
-alanylation of teichoid acids), and the
mprF
gene (
l
-lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a
dltA
mutant showed that Dlt plays a major role in AMP resistance in
L. casei
. This is the first report on the regulation of the response of
L. casei
to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism.
Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli
Authors: Falcioni Francesco , Blank Lars M. , Frick Oliver , Karau Andreas , Bühler Bruno , Schmid Andreas 
Published: March 2013

ABSTRACT

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant
Escherichia coli
expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter
p4h
mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of
putA
and
putP
, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.
Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation
Authors: Yang Haiquan , Lu Xinyao , Liu Long , Li Jianghua , Shin Hyun-dong , Chen Rachel R. , Du Guocheng , Chen Jian 
Published: March 2013

ABSTRACT

In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline α-amylase (AmyK) from
Alkalimonas amylolytica
. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (
k
cat
) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK:
k
cat
/

K
m

increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ∼2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H
2
O
2
, compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes.
Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin
Authors: Ianiri G. , Idnurm A. , Wright S. A. I. , Durán-Patrón R. , Mannina L. , Ferracane R. , Ritieni A. , Castoria R. 
Published: March 2013

ABSTRACT

Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum
Pucciniomycotina
have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study,
Sporobolomyces
sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (
Z
)
-
ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of
Escherichia coli
to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of
Saccharomyces cerevisiae YCK2
,
PAC2
,
DAL5
, and
VPS8
. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.
Novel Rod-Shaped Magnetotactic Bacteria Belonging to the Class Alphaproteobacteria
Authors: Zhang Wen-Yan , Zhou Ke , Pan Hong-Miao , Du Hai-Jian , Chen Yi-Ran , Zhang Rui , Ye Wanneng , Lu Chaojing , Xiao Tian , Wu Long-Fei 
Published: March 2013

ABSTRACTNovel large, rod-shaped magnetotactic bacteria (MTB) were discovered in intertidal sediments of the Yellow Sea, China. They biomineralized more than 300 rectangular magnetite magnetosomes per cell. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that they are affiliated with theAlphaproteobacteriaand may represent a new genus of MTB.
Occurrence, Genetic Diversity, and Persistence of Enterococci in a Lake Superior Watershed
Authors: Ran Qinghong , Badgley Brian D. , Dillon Nicholas , Dunny Gary M. , Sadowsky Michael J. 
Published: March 2013

ABSTRACT

In 2012, the U.S. EPA suggested that coastal and Great Lakes states adopt enterococci as an alternative indicator for the monitoring of recreational water quality. Limited information, however, is available about the presence and persistence of enterococci in Lake Superior. In this study, the density, species composition, and persistence of enterococci in sand, sediment, water, and soil samples were examined at two sites in a Lake Superior watershed from May to September over a 2-year period. The genetic diversity of
Enterococcus faecalis
isolates collected from environmental samples was also studied by using the horizontal, fluorophore-enhanced repetitive PCR DNA fingerprinting technique. Results obtained by most-probable-number analyses indicated that enterococci were present in 149 (94%) of 159 samples and their densities were generally higher in the summer than in the other months examined. The
Enterococcus
species composition displayed spatial and temporal changes, with the dominant species being
E. hirae
,
E. faecalis
,
E. faecium
,
E. mundtii
, and
E. casseliflavus
. DNA fingerprint analyses indicated that the
E. faecalis
population in the watershed was genetically diverse and changed spatially and temporally. Moreover, some DNA fingerprints reoccurred over multiple sampling events. Taken together, these results suggest that some enterococci are able to persist and grow in the Lake Superior watershed, especially in soil, for a prolonged time after being introduced.
Comparison of the Compositions of the Stool Microbiotas of Infants Fed Goat Milk Formula, Cow Milk-Based Formula, or Breast Milk
Authors: Tannock Gerald W. , Lawley Blair , Munro Karen , Gowri Pathmanathan Siva , Zhou Shao J. , Makrides Maria , Gibson Robert A. , Sullivan Thomas , Prosser Colin G. , Lowry Dianne , Hodgkinson Alison J. 
Published: March 2013

ABSTRACTThe aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences andLachnospiraceaesequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. TheLachnospiraceaewere mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milk-fed babies.Bifidobacteriaceaewere abundant in the microbiotas of infants in all three groups.Bifidobacterium longum,Bifidobacterium breve, andBifidobacterium bifidumwere the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate betweenB. longumsubsp.longumandB. longumsubsp.infantisand was used to test stool samples.B. longumsubsp.infantiswas seldom present in stools, even of breast milk-fed babies. The presence ofB. bifidumin the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances ofBifidobacteriaceae. WhenBifidobacteriaceaeabundance was low,Lachnospiraceaeabundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained.
Bioengineering of Bacterial Polymer Inclusions Catalyzing the Synthesis of
N
-Acetylneuraminic Acid
Authors: Hooks David O. , Blatchford Paul A. , Rehm Bernd H. A. 
Published: March 2013

ABSTRACT

N
-Acetylneuraminic acid is produced by alkaline epimerization of
N
-acetylglucosamine to
N
-acetylmannosamine and then subsequent condensation with pyruvate catalyzed by free
N
-acetylneuraminic acid aldolase. The high-alkaline conditions of this process result in the degradation of reactants and products, while the purification of free enzymes to be used for the synthesis reaction is a costly process. The use of
N
-acetylglucosamine 2-epimerase has been seen as an alternative to the alkaline epimerization process. In this study, these two enzymes involved in
N
-acetylneuraminic acid production were immobilized to biopolyester beads
in vivo
in a one-step, cost-efficient process of production and isolation. Beads with epimerase-only, aldolase-only, and combined epimerase/aldolase activity were recombinantly produced in
Escherichia coli
. The enzymatic activities were 32 U, 590 U, and 2.2 U/420 U per gram dry bead weight, respectively. Individual beads could convert 18% and 77% of initial GlcNAc and ManNAc, respectively, at high substrate concentrations and near-neutral pH, demonstrating the application of this biobead technology to fine-chemical synthesis. Beads establishing the entire
N
-acetylneuraminic acid synthesis pathway were able to convert up to 22% of the initial
N
-acetylglucosamine after a 50-h reaction time into
N
-acetylneuraminic acid.
Metabolic Engineering for
l
-Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli
Authors: Hayashi Mikiro , Tabata Kazuhiko 
Published: March 2013

ABSTRACT

An
l
-glutamine-overproducing mutant of an
Escherichia coli
K-12-derived strain was selected from randomly mutagenized cells in the course of
l
-alanyl-
l
-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for
l
-glutamine overproduction in this mutant. Three mutations were identified that are related to the
l
-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region of
yeiG
and two nonsynonymous mutations in
gyrA
(Gly821Ser and Asp830Asn). Expression of
yeiG
, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in
gyrA
, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (
topA
) and topoisomerase IV (
parC
and
parE
), an increase in
l
-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in
l
-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an
l
-glutamine-hyperproducing strain by attenuating cellular
l
-glutamine degradation activity. Although the reconstituted mutant (with
yeiG
together with
gyrA
) produced 200 mM
l
-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM
l
-glutamine.
Evaluating the Assignment of
alkB
Terminal Restriction Fragments and Sequence Types to Distinct Bacterial Taxa
Authors: Giebler Julia , Wick Lukas Y. , Schloter Michael , Harms Hauke , Chatzinotas Antonis 
Published: March 2013

ABSTRACT

Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple
alkB
gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by
alkB
terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and
alkB
gene sequences were highly inconsistent.
Structural Analysis of Succinoglycan Oligosaccharides from Sinorhizobium meliloti Strains with Different Host Compatibility Phenotypes
Authors: Simsek Senay , Wood Karl , Reuhs Bradley L. 
Published: March 2013

ABSTRACT

Sinorhizobium meliloti
NRG247 has a Fix
+
phenotype on
Medicago truncatula
A20 and is Fix
−
on
M. truncatula
A17, and the phenotype is reversed with
S. meliloti
NRG185. As the succinoglycan was shown to impact host specificity, an analysis of the succinoglycan oligosaccharides produced by each strain was conducted. The symbiotically active
s
uccinoglycan
t
rimeric
o
ligosaccharides (STOs) from the two
S. meliloti
strains were compared by chromatography and mass spectrometry, and the analysis of the
S. meliloti
NRG247 oligosaccharides showed that this strain produces an abundance of STO trimer 1 (T1), containing no succinate (i.e., three nonsuccinylated repeats), yet the low-molecular-weight pool contained no nonsuccinylated monomers (potential repeats). This showed that STO T1 is likely to be the active signal on
M. truncatula
A20 and that the biosynthesis of the STOs is not a random polymerization of the monomer population. The results also suggest that the fully succinylated STO T7 is required for the infection of
M. truncatula
A17.
PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA
Authors: Tammam Stephanie , Sampaleanu Liliana M. , Koo Jason , Manoharan Kumararaaj , Daubaras Mark , Burrows Lori L. , Howell P. Lynne 
Published: March 2013

ABSTRACT

Pseudomonas aeruginosa
type IV pili (T4P) are virulence factors that promote infection of cystic fibrosis and immunosuppressed patients. As the absence of T4P impairs colonization, they are attractive targets for the development of novel therapeutics. Genes in the
pilMNOPQ
operon are important for both T4P assembly and a form of bacterial movement, called twitching motility, that is required for pathogenicity. The type II membrane proteins, PilN and PilO, dimerize via their periplasmic domains and anchor this complex in the inner membrane. Our earlier work showed that PilNO binds PilP, a periplasmic lipoprotein (S. Tammam, L. M. Sampaleanu, J. Koo, P. Sundaram, M. Ayers, P. A. Chong, J. D. Forman-Kay, L. L. Burrows, and P. L. Howell, Mol. Microbiol.
82:
1496–1514, 2011). Here, we show that PilP interacts with the N0 segment of the outer membrane secretin PilQ via its C-terminal domain, and that the N-terminal cytoplasmic tail of PilN binds to the actin-like protein PilM, thereby connecting all cellular compartments via the PilMNOPQ protein interaction network. We show that PilA, the major pilin subunit, interacts with PilNOPQ. The results allow us to propose a model whereby PilA makes extensive contacts with the transenvelope complex, possibly to increase local concentrations of PilA monomers for polymerization. The PilNOP complex could provide a stable anchor in the inner membrane, while the PilMNOPQ transenvelope complex facilitates transit of the pilus through the periplasm and clamps the pilus in the cell envelope. The PilMN interaction is proposed to be responsible for communicating signals from the cytoplasmic to periplasmic components of this complex macromolecular machine.
S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation
Authors: Wong Alexander G. , McBurney Kristina L. , Thompson Katharine J. , Stickney Leigh M. , Mackie George A. 
Published: March 2013

ABSTRACT

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient
pnp
::
lacZ
fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using
in vitro
band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent

K
D

values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.
The tRNA Thiolation Pathway Modulates the Intracellular Redox State in Escherichia coli
Authors: Nakayashiki Toru , Saito Natsumi , Takeuchi Rikiya , Kadokura Hiroshi , Nakahigashi Kenji , Wanner Barry L. , Mori Hirotada 
Published: March 2013

ABSTRACT

We have performed a screening of hydroxyurea (HU)-sensitive mutants using a single-gene-deletion mutant collection in
Escherichia coli
K-12. HU inhibits ribonucleotide reductase (RNR), an enzyme that catalyzes the formation of deoxyribonucleotides. Unexpectedly, seven of the mutants lacked genes that are required for the incorporation of sulfur into a specific tRNA modification base, 5-methylaminomethyl-2-thiouridine (mnm
5
s
2
U), via persulfide relay. We found that the expression of RNR in the mutants was reduced to about one-third both in the absence and presence of HU, while sufficient deoxynucleoside triphosphate (dNTP) was maintained in the mutants in the absence of HU but a shortage occurred in the presence of HU.
Trans
-supply of an RNR R2 subunit rescued the HU sensitivity of these mutants. The mutants showed high intracellular ATP/ADP ratios, and overexpression of Hda, which catalyzes the conversion of DnaA-ATP to DnaA-ADP, rescued the HU sensitivity of the mutants, suggesting that DnaA-ATP represses RNR expression. The high intracellular ATP/ADP ratios were due to high respiration activity in the mutants. Our data suggested that intracellular redox was inclined toward the reduced state in these mutants, which may explain a change in RNR activity by reduction of the catalytically formed disulfide bond and high respiration activity by the NADH reducing potential. The relation between persulfide relay and intracellular redox is discussed.
Gene PA2449 Is Essential for Glycine Metabolism and Pyocyanin Biosynthesis in Pseudomonas aeruginosa PAO1
Authors: Lundgren Benjamin R. , Thornton William , Dornan Mark H. , Villegas-Peñaranda Luis Roberto , Boddy Christopher N. , Nomura Christopher T. 
Published: March 2013

ABSTRACT

Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in
Pseudomonas aeruginosa
PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in
P. aeruginosa
PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria.