bluuebunny/crossref_metadata_2025_split huggingface dataset:
Customization of Product Technology and International New Product Success: Mediating Effects of New Product Development and Rollout Timeliness
Authors: Chryssochoidis George M. , Wong Veronica 
Published: October 2003

Offering a standardized product for different country markets may enable companies to accomplish fast product development and multicountry rollout, whereas also enjoying substantial cost benefits. However, not all manufacturers serving multicountry markets can adopt a standardized product strategy. Where technological requirements, standards, and approval procedures vary substantially across countries, manufacturers invariably must adapt the product's technology to fit individual country requirements. Extensive customization may lead to longer new product development and rollout times and increase the likelihood of delays in the entire project, hence adversely affecting overall new product outcome.This study examines the relationships between product technology customization, the timeliness in completion of both the new product development effort and international market launches, and new product success. The study that reports on new product launches across European markets, is based on personal interviews with senior managers in 30 multinational companies. The authors show that timeliness in new product development and timeliness in rolling out the new product into different country markets mediate the link between product technology customization and overall new product success. Customization of product technology increases the likelihood of delays in the completion of new product development projects and multicountry rollout. Additionally, the timeliness in new product development mediates the relationship between product technology customization and timeliness in international new product rollout. This means that if the NPD project runs behind schedule, a fault‐free multicountry rollout program becomes increasingly unlikely, as problems encountered during product development spillover into the rollout program.The results imply that international product managers must assign greater priority to assessing the relative advantages of customizing new product technology and to consider the timing implications for both the NPD effort and subsequent rollout. Managers must set realistic schedules and allocate sufficient resources to ensure both tasks can be accomplished within planned time scales. Finally, managers should not underestimate the complexities and time involved in customizing new product technologies, including the completion of disparate country technical approval procedures.
Production of High-Quality Particulate Methane Monooxygenase in High Yields from
Methylococcus capsulatus
(Bath) with a Hollow-Fiber Membrane Bioreactor
Authors: Yu Steve S.-F. , Chen Kelvin H.-C. , Tseng Mandy Y.-H. , Wang Yane-Shih , Tseng Chiu-Feng , Chen Yu-Ju , Huang Ded-Shih , Chan Sunney I. 
Published: October 2003

ABSTRACT

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from
Methylococcus capsulatus
(Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 μM. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ∼13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl β-
d
-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an αβγ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.
Redefining the Role of
psr
in β-Lactam Resistance and Cell Autolysis of
Enterococcus hirae
Authors: Sapunaric Frédéric , Franssen Christine , Stefanic Patrick , Amoroso Ana , Dardenne Olivier , Coyette Jacques 
Published: October 2003

ABSTRACT

The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the β-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of
Enterococcus hirae
was investigated by disruption or substitution of the corresponding
pbp5
and
psr
genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of
E. hirae
to β-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of
psr
in ATCC 9790 or complementation of R40 cells with
psr
did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the
psr
gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in β-lactam resistance or in the regulation of cell autolysis in
E. hirae
.
Cloning and Characterization of Archaeal Type I Signal Peptidase from
Methanococcus voltae
Authors: Ng Sandy Y. M. , Jarrell Ken F. 
Published: October 2003

ABSTRACT

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in
Methanococcus voltae
was cloned and overexpressed in
Escherichia coli
, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the
M. voltae
S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With
M. voltae
membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the
M. voltae
S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although
E. coli
signal peptidase was able to correctly process the signal peptide during overexpression of the
M. voltae
S-layer protein in vivo, the contribution of the
E. coli
signal peptidase to cleavage of the substrate in the in vitro assay was minimal since
E. coli
membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for
E. coli
signal peptidase I), efficient processing of the substrate was observed only when the
E. coli
membranes contained overexpressed
M. voltae
signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.
Osmotic Adaptation of
Thermus thermophilus
RQ-1: Lesson from a Mutant Deficient in Synthesis of Trehalose
Authors: Silva Zélia , Alarico Susana , Nobre Ana , Horlacher Reinhold , Marugg Joey , Boos Winfried , Mingote Ana I. , da Costa Milton S. 
Published: October 2003

ABSTRACT

Strains of
Thermus thermophilus
accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from
T. thermophilus
strain RQ-1 carrying
otsA
(encoding trehalose-phosphate synthase [TPS]),
otsB
(encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5′ end of
treS
(trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including
treS
) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in
T. thermophilus
RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (
otsA
) and TPP (
otsB
) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of
otsA
and
otsB
reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in
T. thermophilus
RQ-1.
Restricted Translocation across the Cell Wall Regulates Secretion of the Broad-Range Phospholipase C of
Listeria monocytogenes
Authors: Snyder Aleksandra , Marquis Hélène 
Published: October 2003

ABSTRACT

The virulence of
Listeria monocytogenes
is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain. Mpl, a secreted metalloprotease of
Listeria
, is involved in the proteolytic activation of PC-PLC. We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH. pH-regulated release of active PC-PLC is Mpl dependent. To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated. Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight. Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall. Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol. These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.
An Archaeal Chromosomal Autonomously Replicating Sequence Element from an Extreme Halophile,Halobacteriumsp. Strain NRC-1
Authors: Berquist Brian R. , DasSarma Shiladitya 
Published: October 2003

ABSTRACTWe report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophileHalobacteriumstrain NRC-1. The putative replication origin was identified by association with theorc7gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of theorc7gene translational start, plus theorc7gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaealorcgenes (orc6andorc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream oforc7is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure nearorc7homologs in the genomic sequences of two other halophiles,Haloarcula marismortuiandHaloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked toorc7homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number oforc1-cdc6homologs identified in the genomes of all haloarchaea to date.
ThesloABCROperon ofStreptococcus mutansEncodes an Mn and Fe Transport System Required for Endocarditis Virulence and Its Mn-Dependent Repressor
Authors: Paik Sehmi , Brown Arunsri , Munro Cindy L. , Cornelissen Cynthia Nau , Kitten Todd 
Published: October 2003

ABSTRACTStreptococcus mutansbelongs to the viridans group of oral streptococci, which is the leading cause of endocarditis in humans. The LraI family of lipoproteins in viridans group streptococci and other bacteria have been shown to function as virulence factors, adhesins, or ABC-type metal transporters. We previously reported the identification of theS. mutansLraI operon,sloABCR, which encodes components of a putative metal uptake system composed of SloA, an ATP-binding protein, SloB, an integral membrane protein, and SloC, a solute-binding lipoprotein, as well as a metal-dependent regulator, SloR. We report here the functional analysis of this operon. By Western blotting, addition of Mn to the growth medium repressed SloC expression in a wild-type strain but not in asloRmutant. Other metals tested had little effect. Cells were also tested for aerobic growth in media stripped of metals then reconstituted with Mg and either Mn or Fe. Fe at 10 μM supported growth of the wild-type strain but not of asloAorsloCmutant. Mn at 0.1 μM supported growth of the wild-type strain andsloRmutant but not ofsloAorsloCmutants. The combined results suggest that the SloABC proteins transport both metals, although the SloR protein represses this system only in response to Mn. These conclusions are supported by55Fe uptake studies with Mn as a competitor. Finally, asloAmutant demonstrated loss of virulence in a rat model of endocarditis, suggesting that metal transport is required for endocarditis pathogenesis.
Mechanism ofPseudomonas aeruginosaRhlR Transcriptional Regulation of therhlABPromoter
Authors: Medina Gerardo , Juárez Katy , Valderrama Brenda , Soberón-Chávez Gloria 
Published: October 2003

ABSTRACTPseudomonas aeruginosacontains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of therhlABoperon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in therhlABregulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression ofP. aeruginosavirulence-associated traits.
Constitutive Activation of the
Escherichia coli
Pho Regulon Upregulates
rpoS
Translation in an Hfq-Dependent Fashion
Authors: Ruiz Natividad , Silhavy Thomas J. 
Published: October 2003

ABSTRACT

Regulation of the σ factor RpoS occurs at the levels of transcription, translation, and protein stability activity, and it determines whether
Escherichia coli
turns on or off the stationary-phase response. To better understand the regulation of RpoS, we conducted genetic screens and found that mutations in the
pst
locus cause accumulation of RpoS during exponential growth. The
pst
locus encodes for the components of the high-affinity transport system for inorganic phosphate (P
i
), which is involved in sensing P
i
levels in the environment. When the Pst transporter is compromised (either by mutation or by P
i
starvation), the two-component system PhoBR activates the transcription of the Pho regulon, a subset of genes that encode proteins for transporting and metabolizing alternative phosphate sources. Our data show that strains carrying mutations which constitutively activate the Pho regulon have increased
rpoS
translation during exponential growth. This upregulation of
rpoS
translation is Hfq dependent, suggesting the involvement of a small regulatory RNA (sRNA). The transcription of this yet-to-be-identified sRNA is regulated by the PhoBR two-component system.
Transcription Regulation by Tandem-Bound FNR at
Escherichia coli
Promoters
Authors: Barnard Anne M. L. , Green Jeffrey , Busby Stephen J. W. 
Published: October 2003

ABSTRACT

FNR is an
Escherichia coli
transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the
melR
promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the
yfiD
promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.
Characterization of the Two
Mycobacterium tuberculosis recA
Promoters
Authors: Gopaul Krishna K. , Brooks Patricia C. , Prost Jean-François , Davis Elaine O. 
Published: October 2003

ABSTRACT

The
recA
gene of
Mycobacterium tuberculosis
is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to
Escherichia coli
σ
70
−35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to
recA
expression and compared their kinetics of induction following DNA damage.
Rgg Coordinates Virulence Factor Synthesis and Metabolism inStreptococcus pyogenes
Authors: Chaussee Michael S. , Somerville Greg A. , Reitzer Larry , Musser James M. 
Published: October 2003

ABSTRACTStreptococcus pyogenesis a human-specific pathogen that relies on its host for metabolic substrates. Rgg-like proteins constitute a family of transcriptional regulators present in several gram-positive bacteria. InS. pyogenes, Rgg influences the expression of several virulence-associated proteins localized to the cell wall and extracellular environment. Secreted enzymes may degrade host macromolecules, thereby liberating metabolic substrates. To determine if Rgg regulation of exoprotein expression is associated with altered metabolism, the catabolic activities ofS. pyogenesstrain NZ131 (serotype M49) and an isogenicrggmutant strain were analyzed during growth with complex and defined media. As expected, the wild-type strain preferentially used glucose and produced lactic acid during the exponential phase of growth. In contrast, therggmutant fermented arginine in the exponential phase of growth, even in the presence of glucose. Arginine degradation was associated with a neutral culture pH and excretion of NH3and ornithine. Arginine, serine, and asparagine were depleted from mutant cultures during growth. The addition of arginine and serine to culture media increased the growth yield and NH3production of mutant but not wild-type cultures. Addition of asparagine had no effect on the growth yield of either strain. Altered metabolism of arginine and serine in the mutant was associated with increased transcript levels of genes encoding arginine deiminase and a putative serine dehydratase. Thus, Rgg coordinates virulence factor synthesis and catabolic activity and may be important in the pathogen's adaptation to changes in the availability of metabolic substrates.
The Iron-Responsive Regulator Fur Is Transcriptionally Autoregulated and Not Essential inNeisseria meningitidis
Authors: Delany Isabel , Ieva Raffaele , Alaimo Cristina , Rappuoli Rino , Scarlato Vincenzo 
Published: October 2003

ABSTRACTFur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. Thefurgene inNeisseria meningitidishas been described as an essential gene that may regulate a broad array of genes. We succeeded in obtaining anN. meningitidismutant with thefurgene knocked out and used it to undertake studies offur-mediated iron regulation. We show that expression of both Fur and the transferrin binding protein Tbp2 is iron regulated and demonstrate that this regulation is Fur mediated for the Tbp2 protein. Footprinting analysis revealed that Fur binds to two distinct sites upstream of its coding region with different affinities and that these binding sites overlap two promoters that differentially control transcription of thefurgene in response to iron. The presence of two independently regulatedfurpromoters may allow meningococcus to fine-tune expression of this regulator controlling iron homeostasis, possibly during infection.