bluuebunny/crossref_metadata_2025_split huggingface dataset:
Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of
Bacillu
s Spores
Authors: Hathout Yetrib , Setlow Barbara , Cabrera-Martinez Rosa-Martha , Fenselau Catherine , Setlow Peter 
Published: February 2003

ABSTRACT

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different
Bacillus
spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor α/β- and γ-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (
B. cereus
T and
B. thuringiensis
subsp.
Kurstaki
) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.
A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast
Pichia pastoris
Authors: Hanquier José , Sorlet Yannick , Desplancq Dominique , Baroche Laurence , Ebtinger Marc , Lefèvre Jean-François , Pattus Franc , Hershberger Charles L. , Vertès Alain A. 
Published: February 2003

ABSTRACT

We produced bovine trypsinogen in the yeast
Pichia pastoris
. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong
aox1
promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.
Effects of Alternative Dietary Substrates on Competition between Human Colonic Bacteria in an Anaerobic Fermentor System
Authors: Duncan Sylvia H. , Scott Karen P. , Ramsay Alan G. , Harmsen Hermie J. M. , Welling Gjalt W. , Stewart Colin S. , Flint Harry J. 
Published: February 2003

ABSTRACTDuplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion ofBacteroidesand a lower proportion of gram-positive anaerobes related toFaecalibacterium prausnitzii,Ruminococcus flavefaciens-Ruminococcus bromii,Eubacterium rectale-Clostridium coccoides, andEubacterium cylindroidesthan the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related toR. flavefaciens-R. bromiiandE. cylindroides.The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. TheRoseburia-related strain A2-183Fwas able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast,Roseburia intestinalisL1-82RandEubacteriumsp. strain A2-194Rsurvived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194Rwas stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.
Monitoring the Production of Aflatoxin B
1
in Wheat by Measuring the Concentration of
nor-1
mRNA
Authors: Mayer Zsuzsanna , Färber Paul , Geisen Rolf 
Published: February 2003

ABSTRACT

A real-time reverse transcription-PCR system has been used to monitor the expression of an aflatoxin biosynthetic gene of
Aspergillus flavus
in wheat. Therefore, total RNA was isolated from infected wheat samples, reverse transcribed and subjected to real-time PCR. In parallel all samples were analyzed by high-pressure liquid chromatography for aflatoxin B
1
production. The primer-probe system of the real-time PCR was targeted against
nor-1
, a gene of the aflatoxin biosynthetic pathway. By application of this method the
nor-1
transcription was quantified during the course of incubation. After 4 days of incubation
nor-1
mRNA could be detected for the first time. The amount of
nor-1
mRNA increased rapidly, and the maximum was achieved after 6 days. Then, starting very slowly, the mRNA was degraded until day 8, and this was followed by a very fast degradation, reaching nondetectable levels at days 9 and 10. First traces of aflatoxin B
1
could be detected between the 5th and 6th day of incubation. The aflatoxin concentration reached its maximum after 9 days of incubation and remained constant for the whole period of observation. To ensure that differences in the
nor-1
mRNA concentration were due to different expression levels, the expression of the constitutively expressed β-tubulin gene (
benA56
) has also been monitored. The expression of
benA56
remained constant during the whole incubation time. As a parameter for fungal growth, the number of
nor-1
gene copies was determined during the course of incubation. The numbers of
nor-1
gene copies increased at the beginning of the incubation and reached a plateau at day 5. They correlate well with the viable counts albeit at a higher level.
Involvement of Calcium/Calmodulin Signaling in Cercosporin Toxin Biosynthesis byCercosporanicotianae
Authors: Chung Kuang-Ren 
Published: February 2003

ABSTRACTCercosporin is a non-host-selective, perylenequinone toxin produced by many phytopathogenicCercosporaspecies. The involvement of Ca2+/calmodulin (CaM) signaling in cercosporin biosynthesis was investigated by using pharmacological inhibitors. The results suggest that maintaining endogenous Ca2+homeostasis is required for cercosporin biosynthesis inCercosporanicotianae. The addition of excess Ca2+to the medium slightly increased fungal growth but resulted in a reduction in cercosporin production. The addition of Ca2+chelators [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] also reduced cercosporin production. Ca2+channel blockers exhibited a strong inhibition of cercosporin production only at higher concentrations (>2 mM). Cercosporin production was reduced greatly by Ca2+ionophores (A23187 and ionomycin) and internal Ca2+blocker [3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester]. Phospholipase C inhibitors (lithium, U73122, and neomycin) led to a concentration-dependent inhibition of cercosporin biosynthesis. Furthermore, the addition of CaM inhibitors (compound 48/80, trifluoperazine, W-7, and chlorpromazine) also markedly reduced cercosporin production. In contrast to W-7, W-5, with less specificity for CaM, led to only minor inhibition of cercosporin production. The inhibitory effects of Ca2+/CaM inhibitors were partially or completely reversed by the addition of external Ca2+. As assessed with Fluo-3/AM (a fluorescent Ca2+indicator), the Ca2+content in the cytoplasm decreased significantly when fungal cultures were grown in a medium containing Ca2+/CaM antagonists, confirming the specificity of those Ca2+/CaM antagonists inC.nicotianae. Taken together, the results suggest that Ca2+/CaM signal transduction may play a pivotal role in cercosporin biosynthesis inC.nicotianae.
Development of
Sinorhizobium meliloti
Pilot Macroarrays for Transcriptome Analysis
Authors: Bergès Hélène , Lauber Emmanuelle , Liebe Carine , Batut Jacques , Kahn Daniel , de Bruijn Frans J. , Ampe Frédéric 
Published: February 2003

ABSTRACT

In order to prepare for whole-genome expression analysis in
Sinorhizobium meliloti
, pilot DNA macroarrays were designed for 34 genes of known regulation. The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5′ end of the primers, and the method of RNA labeling. Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method. The sizes of PCR products were also found to be important, whereas the influence of 5′ tags was minimal. The variability between replicated spots on a membrane was found to be low. These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression.
Characterization of a New Operon,as-48EFGH, from theas-48Gene Cluster Involved in Immunity to Enterocin AS-48
Authors: Diaz Marta , Valdivia Eva , Martínez-Bueno Manuel , Fernández Matilde , Soler-González Andrés Santos , Ramírez-Rodrigo Hilario , Maqueda Mercedes 
Published: February 2003

ABSTRACTEnterocin AS-48 is a cyclic peptide produced byEnterococcus faecalisS-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, theas-48Astructural gene andas-48B,as-48C,as-48C1,as-48D, andas-48D1genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in theBglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs,as-48Gandas-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5disruption experiments in the last genes,as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. Theas-48EFGHoperon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T2and T3, in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T3could be regulated, because in JH2-2(pAM401EH) transformants, T3was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for theas-48D1gene for immunity against AS-48.
Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant
Escherichia
coli
Strains
Authors: Ignatova Z. , Mahsunah A. , Georgieva M. , Kasche V. 
Published: February 2003

ABSTRACT

Using periplasmic penicillin amidase (PA) from
Escherichia
coli
ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant
E
.
coli
expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (

k
d

) and transport (

k
t

) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain
E
.
coli
BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants

k
d

and

k
t

. The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different
E
.
coli
host strains. Furthermore, we established that simultaneous coexpression of the OmpT
pac
gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the
kil
gene (killing protein). The primary effects of the
kil
gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.
Aminopeptidase C of
Aspergillus niger
Is a Novel Phenylalanine Aminopeptidase
Authors: Basten Daniëlle E. J. W. , Dekker Peter J. T. , Schaap Peter J. 
Published: February 2003

ABSTRACT

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from
Aspergillus niger
(CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine
para
-nitroanilide (F-
p
NA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either
p
NA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze
N
-acetylalanine-
p
NA, a substrate for acyl-peptide hydrolases.
Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various
Streptomyces avermitilis
Strains
Authors: Hwang Yong-Soon , Kim Eung-Soo , Biró Sándor , Choi Cha-Yong 
Published: February 2003

ABSTRACT

To isolate a gene for stimulating avermectin production, a genomic library of
Streptomyces avermitilis
ATCC 31267 was constructed in
Streptomyces lividans
TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type
S. avermitilis
was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of
Streptomyces coelicolor
A3(2). The
orfX
gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of
orfX
revealed that this gene was intrinsically involved in the stimulation of avermectin production in
S. avermitilis
.
Molecular Characterization of
tet
(M) Genes in
Lactobacillus
Isolates from Different Types of Fermented Dry Sausage
Authors: Gevers Dirk , Danielsen Morten , Huys Geert , Swings Jean 
Published: February 2003

ABSTRACT

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc
r
) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the
tet
genes, conferring tetracycline resistance, that they carry. With the (GTG)
5
-PCR DNA fingerprinting technique, the Tc
r
lactic acid bacterial isolates were identified as
Lactobacillus plantarum
,
L. sakei
subsp.
carnosus
,
L. sakei
subsp.
sakei
,
L. curvatus
, and
L. alimentarius
and typed to the intraspecies level. For a selection of 24 Tc
r
lactic acid bacterial isolates displaying unique (GTG)
5
-PCR fingerprints,
tet
genes were determined by means of PCR, and only
tet
(M) was detected. Restriction enzyme analysis with
Acc
I and
Sca
I revealed two different
tet
(M) allele types. This grouping was confirmed by partial sequencing of the
tet
(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with
tet
(M) genes previously reported in
Staphylococcus aureus
MRSA 101 and in
Neisseria meningitidis
, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained
tet
(M)-carrying plasmids. In addition to the
tet
(M) gene, one isolate also contained an
erm
(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the
tet
(M) genes were not located on transposons of the Tn
916
/Tn
1545
family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse
Lactobacillus
strains from different types of fermented meat products can be a host for plasmid-borne
tet
genes.
Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments
Authors: Ji Pingsheng , Wilson Mark 
Published: February 2003

ABSTRACT

Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents
Pseudomonas syringae
Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved. Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves. Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed. This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of
P. syringae
in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.
Enhanced Production of Recombinant Proteins in
Escherichia coli
by Filamentation Suppression
Authors: Jeong Ki Jun , Lee Sang Yup 
Published: February 2003

ABSTRACT

During growth of high-cell-density cultures of
Escherichia coli
, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the
E. coli ftsA
and
ftsZ
genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the
ftsA
and
ftsZ
genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.
High Rate of Uptake of Organic Nitrogen Compounds by
Prochlorococcus
Cyanobacteria as a Key to Their Dominance in Oligotrophic Oceanic Waters
Authors: Zubkov Mikhail V. , Fuchs Bernhard M. , Tarran Glen A. , Burkill Peter H. , Amann Rudolf 
Published: February 2003

ABSTRACT

Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported. About 33% of the total bacterioplankton turnover of amino acids, determined with a representative [
35
S]methionine precursor and flow sorting, can be assigned to
Prochlorococcus
spp. and 3% can be assigned to
Synechococcus
spp. in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively. This finding may provide a mechanism for
Prochlorococcus
' competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop.
Studies on the Mode of Action of Reutericyclin
Authors: Gänzle Michael G. , Vogel Rudi F. 
Published: February 2003

ABSTRACT
The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential.
Cloning and Expression Analysis of the
pcbAB-pcbC
β-Lactam Genes in the Marine Fungus
Kallichroma tethys
Authors: Kim Chi-fai , Lee Simon K. Y. , Price Jackie , Jack Ralph W. , Turner Geoffrey , Kong Richard Y. C. 
Published: February 2003

ABSTRACT

Here we report the identification of the β-lactam biosynthesis genes
pcbAB
and
pcbC
from a cosmid genomic DNA library of the marine fungus
Kallichroma tethys.
A BLAST homology search showed that they share high sequence identity with the δ-(
l
-α-aminoadipyl)-
l
-cysteinyl-
d
-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial β-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete
Acremonium chrysogenum
. Expression analysis by reverse transciption-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of
K. tethys
cultures. Complementation of an
Aspergillus nidulans
strain deficient in ACV synthetase suggested that at least
pcbAB
is functional, although attempts to isolate active antibiotic from
K. tethys
were unsuccessful.