bluuebunny/crossref_metadata_2025_split huggingface dataset:
adhA
in
Aspergillus parasiticus
Is Involved in Conversion of 5′-Hydroxyaverantin to Averufin
Authors: Chang Perng-Kuang , Yu Jiujiang , Ehrlich Kenneth C. , Boue Stephen M. , Montalbano Beverly G. , Bhatnagar Deepak , Cleveland Thomas E. 
Published: July 2002

ABSTRACT

Two routes for the conversion of 5′-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5′-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene,
adhA
, from the aflatoxin gene cluster of
Aspergillus parasiticus
SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases—a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD
+
-NADP
+
, and the motif YXXXK, which is found at the active site.
A. parasiticus
SU-1, which produces aflatoxins, has two copies of
adhA
(
adhA1
), whereas
A. parasiticus
SRRC 2043, a strain that accumulates
O
-methylsterigmatocystin (OMST), has only one copy. Disruption of
adhA
in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those
adhA
disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.
Thermostable NADP
+
-Dependent Medium-Chain Alcohol Dehydrogenase from
Acinetobacter
sp. Strain M-1: Purification and Characterization and Gene Expression in
Escherichia coli
Authors: Tani Akio , Sakai Yasuyoshi , Ishige Takeru , Kato Nobuo 
Published: July 2002

ABSTRACT

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by
n
-hexadecane, from
Acinetobacter
sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward
n
-heptanal and could use
n
-alkylaldehydes ranging from C
2
to C
14
and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The
alrA
gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the
alrA
gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in
Escherichia coli
, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.
Spatial Changes in the Bacterial Community Structure along a Vertical Oxygen Gradient in Flooded Paddy Soil Cores
Authors: Lüdemann Heiner , Arth Inko , Liesack Werner 
Published: July 2002

ABSTRACT

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 μM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-μm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the α and β subclasses of
Proteobacteria
as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.
Immunolocalization of Dinitrogenase Reductase Produced by
Klebsiella pneumoniae
in Association with
Zea mays
L
Authors: Chelius Marisa K. , Triplett Eric W. 
Published: July 2002

ABSTRACT

The endophytic lifestyle of
Klebsiella pneumoniae
is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by
K. pneumoniae
strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for
K. pneumoniae
and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.
Characterization and Determination of Origin of Lactic Acid Bacteria from a Sorghum-Based Fermented Weaning Food by Analysis of Soluble Proteins and Amplified Fragment Length Polymorphism Fingerprinting
Authors: Kunene Nokuthula F. , Geornaras Ifigenia , von Holy Alexander , Hastings John W. 
Published: July 2002

ABSTRACT

The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains were
Lactobacillus plantarum
strains, 47 were
Leuconostoc mesenteroides
strains, 25 were
Lactobacillus sake-Lactobacillus curvatus
strains, 17 were
Pediococcus pentosaceus
strains, 13 were
Pediococcus acidilactici
strains, and 7 were
Lactococcus lactis
strains.
L. plantarum
and
L. mesenteroides
strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.
Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions
Authors: Barth S. , Huhn M. , Matthey B. , Klimka A. , Galinski E. A. , Engert A. 
Published: July 2002

ABSTRACT

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of
Escherichia coli
. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of
E. coli
under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.
Long-Chain Aldehyde Dehydrogenase That Participates in
n
-Alkane Utilization and Wax Ester Synthesis in
Acinetobacter
sp. Strain M-1
Authors: Ishige Takeru , Tani Akio , Sakai Yasuyoshi , Kato Nobuo 
Published: July 2002

ABSTRACT

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of
Acinetobacter
sp. strain M-1 cells grown on
n
-hexadecane as a sole carbon source. The gene (
ald1
) was cloned from the chromosomal DNA of the bacterium. The open reading frame of
ald1
was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The
ald1
gene was stably expressed in
Escherichia coli
, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward
n
-alkanals (C
4
to C
14
), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The
ald1
gene was disrupted by homologous recombination on the
Acinetobacter
genome. Although the
ald1
disruptant (
ald1Δ
) strain still had the ability to grow on
n
-hexadecane to some extent, its aldehyde dehydrogenase activity toward
n
-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the
ald1
Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on
n
-alkane and in wax ester formation in
Acinetobacter
sp. strain M-1.
Cold Shock Proteins ofLactococcus lactisMG1363 Are Involved in Cryoprotection and in the Production of Cold-Induced Proteins
Authors: Wouters Jeroen A. , Frenkiel Hélène , de Vos Willem M. , Kuipers Oscar P. , Abee Tjakko 
Published: July 2002

ABSTRACTMembers of the group of 7-kDa cold-shock proteins (CSPs) are the proteins with the highest level of induction upon cold shock in the lactic acid bacteriumLactococcus lactisMG1363. By using double-crossover recombination, twoL. lactisstrains were generated in which genes encoding CSPs are disrupted:L. lactisNZ9000ΔAB lacks the tandemly orientatedcspAandcspBgenes, and NZ9000ΔABE lackscspA, cspB, andcspE. Both strains showed no differences in growth at normal and at low temperatures compared to that of the wild-type strain,L. lactisNZ9000. Two-dimensional gel electrophoresis showed that upon disruption of thecspABgenes, the production of remaining CspE at low temperature increased, and upon disruption ofcspA, cspB, andcspE, the production of CspD at normal growth temperatures increased. Northern blot analysis showed that control is most likely at the transcriptional level. Furthermore, it was established by a proteomics approach that some (non-7-kDa) cold-induced proteins (CIPs) are not cold induced in thecsp-lacking strains, among others the histon-like protein HslA and the signal transduction protein LlrC. This supports earlier observations (J. A. Wouters, M. Mailhes, F. M. Rombouts, W. M. De Vos, O. P. Kuipers, and T. Abee, Appl. Environ. Microbiol. 66:3756–3763, 2000). that the CSPs ofL. lactismight be directly involved in the production of some CIPs upon low-temperature exposure. Remarkably, the adaptive response to freezing by prior exposure to 10°C was significantly reduced in strain NZ9000ΔABE but not in strain NZ9000ΔAB compared to results with wild-type strain NZ9000, indicating a notable involvement of CspE in cryoprotection.
Purification and Properties of a Glucuronan Lyase from
Sinorhizobium meliloti
M5N1CS (NCIMB 40472)
Authors: Da Costa Alexandre , Michaud Philippe , Petit Emmanuel , Heyraud Alain , Colin-Morel Philippe , Courtois Bernard , Courtois Josiane 
Published: July 2002

ABSTRACT

A glucuronan lyase extracted from
Sinorhizobium meliloti
strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn
2+
, Cu
2+
, and Hg
2+
(1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from
S. meliloti
strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.
In Planta Regulation of Extension of an Endophytic Fungus and Maintenance of High Metabolic Rates in Its Mycelium in the Absence of Apical Extension
Authors: Tan Yong Y. , Spiering Martin J. , Scott Vicki , Lane Geoffrey A. , Christensen Michael J. , Schmid Jan 
Published: July 2002

ABSTRACT

The fungus
Neotyphodium lolii
is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass
Lolium perenne,
producing secondary metabolites which enhance the fitness of the association over that of uninfected
L. perenne
. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.
Novel Zn
2+
-Chelating Peptides Selected from a Fimbria-Displayed Random Peptide Library
Authors: Kjærgaard Kristian , Schembri Mark A. , Klemm Per 
Published: July 2002

ABSTRACT

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of
Escherichia coli
which mediate
d
-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the
E. coli
cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of ∼40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn
2+
. By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn
2+
were enriched. None of the isolated sequences showed similarity to known Zn
2+
-binding proteins, indicating that completely novel Zn
2+
-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn
2+
-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.
Influence of Tomato Genotype on Growth of Inoculated and Indigenous Bacteria in the Spermosphere
Authors: Simon Holly M. , Smith Kevin P. , Dodsworth Jeremy A. , Guenthner Bobbi , Handelsman Jo , Goodman Robert M. 
Published: July 2002

ABSTRACT

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent,
Bacillus cereus
UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786–4790, 1999). Here we report results of studies examining the host effect on the support of growth of
Bacillus
and
Pseudomonas
strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of
B. cereus
UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24
B. cereus
strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of
B. cereus
UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains
Pseudomonas fluorescens
2-79 or
Pseudomonas aureofaciens
AB254. Thus, while the host effect on growth extended to all strains of
B. cereus
examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of
B. cereus
UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous
Bacillus
and fluorescent
Pseudomonas
bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous
Bacillus
soil bacteria, while the line that supported growth of inoculated
B. cereus
UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of
B. cereus
UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains—both
Bacillus
and
Pseudomonas
spp.—did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.
Analysis of the Genetic Switch and Replication Region of a P335-Type Bacteriophage with an Obligate Lytic Lifestyle on
Lactococcus lactis
Authors: Madsen Søren M. , Mills David , Djordjevic Gordana , Israelsen Hans , Klaenhammer Todd R. 
Published: July 2002

ABSTRACT

The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage φ31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that φ31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a
lacLM
reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate
Lactococcus lactis
phages. The
cro
gene homologue regulates its own production and is an efficient repressor of
c
I gene expression. No detectable
c
I gene expression could be measured in the presence of
cro. c
I gene expression in the absence of
cro
exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the
cro
gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several
Streptococcus thermophilus
phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of φ31 10
6
-fold when present at high copy number in
trans
provide evidence for identification of the phage origin of replication. Phage φ31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of
Lactococcus
,
Lactobacillus
, and
Streptococcus
were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.
Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains
Authors: Hickey Rita M. , Twomey Denis P. , Ross R. Paul , Hill Colin 
Published: July 2002

ABSTRACT

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (
oriT
). This
oriT
-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10
−4
transconjugants/donor cell from an MG1363 (
recA
mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the
oriT
-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that
oriT
of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.