bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lactococcus lactis
Gene
yjgB
Encodes a γ-
d
-Glutaminyl-
l
-Lysyl- Endopeptidase Which Hydrolyzes Peptidoglycan
Authors: Redko Yulia , Courtin Pascal , Mézange Christine , Huard Carine , Chapot-Chartier Marie-Pierre 
Published: July 2007

ABSTRACT

YjgB is one of five peptidoglycan hydrolases previously identified in
Lactococcus lactis
. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an
L. lactis
strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of
L. lactis
muropeptides between γ-
d
-Gln and
l
-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves γ-
d
-Gln-
l
-Lys bonds in peptide chains of
L. lactis
peptidoglycan.
Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters
Authors: Lee Sung Kuk , Chou Howard H. , Pfleger Brian F. , Newman Jack D. , Yoshikuni Yasuo , Keasling Jay D. 
Published: July 2007

ABSTRACT

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P
BAD
) system that is more compatible with IPTG (isopropyl-β-
d
-1-thiogalactopyranoside) induction of a lactose-inducible (P
lac
) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.
Functional Analysis of the Fructooligosaccharide Utilization Operon in
Lactobacillus paracasei
1195
Authors: Goh Yong Jun , Lee Jong-Hwa , Hutkins Robert W. 
Published: July 2007

ABSTRACT

The
fosABCDXE
operon encodes components of a putative fructose/mannose phosphoenolpyruvate-dependent phosphotransferase system and a β-fructosidase precursor (FosE) that are involved in the fructooligosaccharide (FOS) utilization pathway of
Lactobacillus paracasei
1195. The presence of an N-terminal signal peptide sequence and an LPQAG cell wall anchor motif in the C-terminal region of the deduced FosE precursor amino acid sequence predicted that the enzyme is cell wall associated, indicating that FOS may be hydrolyzed extracellularly. In this study, cell fractionation experiments demonstrated that the FOS hydrolysis activity was present exclusively in the cell wall extract of
L. paracasei
previously grown on FOS. In contrast, no measurable FOS hydrolysis activity was detected in the cell wall extract from the isogenic
fosE
mutant. Induction of β-fructosidase activity was observed when cells were grown on FOS, inulin, sucrose, or fructose but not when cells were grown on glucose. A diauxic growth pattern was observed when cells were grown on FOS in the presence of limiting glucose (0.1%). Analysis of the culture supernatant revealed that glucose was consumed first, followed by the longer-chain FOS species. Transcription analysis further showed that the
fos
operon was expressed only after glucose was depleted in the medium. Expression of
fosE
in a non-FOS-fermenting strain,
Lactobacillus rhamnosus
GG, enabled the recombinant strain to metabolize FOS, inulin, sucrose, and levan.
Water Buffaloes (
Bubalus bubalis
) Identified as an Important Reservoir of Shiga Toxin-Producing
Escherichia coli
in Brazil
Authors: Oliveira Murilo G. , Feitosa Brito José R. , Carvalho Roberta R. , Guth Beatriz E. C. , Gomes Tânia A. T. , Vieira Mônica A. M. , Kato Maria A. M. F. , Ramos Isabel I. , Vaz Tânia M. I. , Irino Kinue 
Published: July 2007

ABSTRACT

The presence of Shiga toxin-producing
Escherichia coli
(STEC) in water buffaloes is reported for the first time in South America. The prevalence of STEC ranged from 0 to 64% depending on the farm. STEC isolates exhibiting the genetic profiles
stx
1
stx
2
ehxA iha saa
and
stx
2
ehxA iha saa
predominated. Of the 20 distinct serotypes identified, more than 50% corresponded to serotypes associated with human diseases.
New Genera of RNA Viruses in Subtropical Seawater, Inferred from Polymerase Gene Sequences
Authors: Culley Alexander I. , Steward Grieg F. 
Published: July 2007

ABSTRACT
Viruses are an integral component of the marine food web, contributing to the disease and mortality of essentially every type of marine life, yet the diversity of viruses in the sea, especially those with RNA genomes, remains very poorly characterized. Isolates of RNA-containing viruses that infect marine plankton are still rare, and the only cultivation-independent surveys of RNA viral diversity reported so far were conducted for temperate coastal waters of British Columbia. Here, we report on our improvements to a previously used protocol to investigate the diversity of marine picorna-like viruses and our results from applying this protocol in subtropical waters. The original protocol was simplified by using direct filtration, rather than tangential flow filtration, to harvest viruses from seawater, and new degenerate primers were designed to amplify a fragment of the RNA-dependent RNA polymerase gene by reverse transcription-PCR from RNA extracted from the filters. Whereas the original protocol was unsuccessful in a preliminary test, the new protocol resulted in amplification of picorna-like virus sequences in every sample of subtropical and temperate coastal seawater assayed. These polymerase sequences formed a diverse, but monophyletic cluster along with other sequences amplified previously from seawater and sequences from isolates infecting marine protists. Phylogenetic analysis suggested that our sequences represent at least five new genera and 24 new species of RNA viruses. These results contribute to our understanding of RNA virus diversity and suggest that picorna-like viruses are a source of mortality for a wide variety of marine protists.
Potential Use of Toxic Thermolabile Proteins To Study Protein Quality Control Systems
Authors: Mizrahi Itzhak , Dagan Michael , Biran Dvora , Ron Eliora Z. 
Published: July 2007

ABSTRACT

SulA is an
Escherichia coli
division inhibitor with a short half-life whose accumulation results in filamentation. Here, we show that SulA is thermally unstable and forms aggregates at elevated temperatures. This property enables the selection of isolates with mutated protein quality control systems.
Discovery of a Novel Styrene Monooxygenase Originating from the Metagenome
Authors: van Hellemond Erik W. , Janssen Dick B. , Fraaije Marco W. 
Published: July 2007

ABSTRACT

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in
Escherichia coli
, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (
S
)-epoxides with excellent enantiomeric excess [e.g., (
S
)-styrene oxide is formed with >99% enantiomeric excess,
ee
] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (
R
)-ethyl phenyl sulfoxide is formed with 92%
ee
]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.
Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of
Neurospora crassa
Authors: Riquelme Meritxell , Bartnicki-García Salomon , González-Prieto Juan Manuel , Sánchez-León Eddy , Verdín-Ramos Jorge A. , Beltrán-Aguilar Alejandro , Freitag Michael 
Published: July 2007

ABSTRACT

The subcellular location and traffic of two selected chitin synthases (CHS) from
Neurospora crassa
, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.
Peroxisomal Peripheral Membrane Protein YlInp1p Is Required for Peroxisome Inheritance and Influences the Dimorphic Transition in the Yeast
Yarrowia lipolytica
Authors: Chang Jinlan , Fagarasanu Andrei , Rachubinski Richard A. 
Published: July 2007

ABSTRACT

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast
Yarrowia lipolytica
. Most peroxisomes are anchored at the periphery of cells of
Y. lipolytica
. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in
Y. lipolytica
. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in
Y. lipolytica
, as cells lacking the Yl
INP1
gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in
Y. lipolytica
.
High-Affinity Interaction between the S-Layer Protein SbsC and the Secondary Cell Wall Polymer ofGeobacillus stearothermophilusATCC 12980 Determined by Surface Plasmon Resonance Technology
Authors: Ferner-Ortner Judith , Mader Christoph , Ilk Nicola , Sleytr Uwe B. , Egelseer Eva M. 
Published: July 2007

ABSTRACTSurface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC31-270] and rSbsC31-443) and the secondary cell wall polymer (SCWP) isolated fromGeobacillus stearothermophilusATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities.
The N-Terminal Propeptide of
Vibrio vulnificus
Extracellular Metalloprotease Is both an Inhibitor of and a Substrate for the Enzyme
Authors: Chang Alan K. , Park Jong Woo , Lee Eun Hee , Lee Jung Sup 
Published: July 2007

ABSTRACT

Vibrio vulnificus
, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP—vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease—were expressed in
Escherichia coli
and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.
Mutations in Residues Involved in Zinc Binding in the Catalytic Site of
Escherichia coli
Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype
Authors: Caillet Joël , Graffe Monique , Eyermann Flore , Romby Pascale , Springer Mathias 
Published: July 2007

ABSTRACT

Escherichia coli
threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA
Thr
isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNA
Thr
by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.
Characterization and Comparison of Biofilm Development by Pathogenic and Commensal Isolates of
Histophilus somni
Authors: Sandal Indra , Hong Wenzhou , Swords W. Edward , Inzana Thomas J. 
Published: July 2007

ABSTRACT

Histophilus somni
(
Haemophilus somnus
) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by
H. somni
prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since
H. somni
is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.
Sulfite Reduction in Mycobacteria
Authors: Pinto Rachel , Harrison Joseph S. , Hsu Tsungda , Jacobs William R. , Leyh Thomas S. 
Published: July 2007

ABSTRACT

Mycobacterium tuberculosis
places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in
Mycobacterium smegmatis
was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from
M. tuberculosis
were determined under strict anaerobic conditions [
k
cat
= 1.0 (±0.1) electron consumed per second, and K

m(SO
3
−2
)

= 27 (±1) μM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (
sirA
, originally misannotated
nirA
) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed
che1
) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe
2+
into siroheme. Deletion of
che1
causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.
SbcCD Regulation and Localization inEscherichia coli
Authors: Darmon Elise , Lopez-Vernaza Manuel A. , Helness Anne C. , Borking Amanda , Wilson Emily , Thacker Zubin , Wardrope Laura , Leach David R. F. 
Published: July 2007

ABSTRACTThe SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. InEscherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of thesbcDCoperon and the cellular localization of the SbcC and SbcD proteins. Transcription of thesbcDCoperon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.
Global Analysis of mRNA Decay in
Halobacterium salinarum
NRC-1 at Single-Gene Resolution Using DNA Microarrays
Authors: Hundt Sonja , Zaigler Alexander , Lange Christian , Soppa Jörg , Klug Gabriele 
Published: July 2007

ABSTRACT

RNA degradation is an important factor in the regulation of gene expression. It allows organisms to quickly respond to changing environmental conditions by adapting the expression of individual genes. The stability of individual mRNAs within an organism varies considerably, contributing to differential amounts of proteins expressed. In this study we used DNA microarrays to analyze mRNA degradation in exponentially growing cultures of the extremely halophilic euryarchaeon
Halobacterium salinarum
NRC-1 on a global level. We determined mRNA half-lives for 1,717 open reading frames, 620 of which are part of known or predicted operons. Under the tested conditions transcript stabilities ranged from 5 min to more than 18 min, with 79% of the evaluated mRNAs showing half-lives between 8 and 12 min. The overall mean half-life was 10 min, which is considerably longer than the ones found in the other prokaryotes investigated thus far. As previously observed in
Escherichia coli
and
Saccharomyces cerevisiae
, we could not detect a significant correlation between transcript length and transcript stability, but there was a relationship between gene function and transcript stability. Genes that are known or predicted to be transcribed in operons exhibited similar mRNA half-lives. These results provide initial insights into mRNA turnover in a euryarchaeon. Moreover, our model organism,
H. salinarum
NRC-1, is one of just two archaea sequenced to date that are missing the core subunits of the archaeal exosome. This complex orthologous to the RNA degrading exosome of eukarya is found in all other archaeal genomes sequenced thus far.