bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Analysis of the Polyhydroxyalkanoate-Specific β-Ketothiolase and Acetoacetyl Coenzyme A Reductase Genes in the Cyanobacterium
Synechocystis
sp. Strain PCC6803
Authors: Taroncher-Oldenburg Gaspar , Nishina Koren , Stephanopoulos Gregory 
Published: July 2002

ABSTRACT

Synechocystis
sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific β-ketothiolase encoded by

phaA
Syn

and an acetoacetyl-coenzyme A (CoA) reductase encoded by

phaB
Syn

. A similarity search of the entire
Synechocystis
genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of
Escherichia coli
with the two genes and the PHA synthase of
Synechocystis
resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster in
Synechocystis
eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific β-ketothiolase and acetoacetyl-CoA reductase of
Synechocystis
and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162–170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin of

phaA
Syn

and

phaB
Syn

in the γ subdivision of the division
Proteobacteria
.
Bacterial Activity in South Pole Snow
Authors: Carpenter Edward J. , Lin Senjie , Capone Douglas G. 
Published: July 2002

ABSTRACT

Large populations (200 to 5,000 cells ml
−1
in snowmelt) of bacteria were present in surface snow and firn from the south pole sampled in January 1999 and 2000. DNA isolated from this snow yielded ribosomal DNA sequences similar to those of several psychrophilic bacteria and a bacterium which aligns closely with members of the genus
Deinococcus
, an ionizing-radiation- and desiccation-resistant genus. We also obtained evidence of low rates of bacterial DNA and protein synthesis which indicates that the organisms were metabolizing at ambient subzero temperatures (−12 to −17°C).
Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System
Authors: Fernández Luis A. , Sola Isabel , Enjuanes Luis , de Lorenzo Víctor 
Published: July 2002

ABSTRACTA simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia colicultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) ofE. colithat specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.
Functional Tn
5393
-Like Transposon in the R Plasmid pRAS2 from the Fish Pathogen
Aeromonas salmonicida
subspecies
salmonicida
Isolated in Norway
Authors: L'Abée-Lund Trine M. , Sørum Henning 
Published: July 2002

ABSTRACT

Tn
5393
c containing
strA-strB
was identified as part of R plasmid pRAS2 from the fish pathogen
Aeromonas salmonicida
subsp.
salmonicida
. This is the first time an intact and active transposon in the Tn
5393
family has been reported in an ecological niche other than an agricultural habitat.
A Novel Genetically Engineered Pathway for Synthesis of Poly(Hydroxyalkanoic Acids) in
Escherichia coli
Authors: Liu Shuang-Jiang , Steinbüchel Alexander 
Published: July 2002

ABSTRACT

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of
Clostridium acetobutylicum
and the two PHA synthase genes (
pha
E and
pha
C) of
Thiocapsa pfennigii
in
Escherichia coli
. The four genes were cloned into the
Bam
HI and
Eco
RI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to
E. coli
JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.
Degradation and Mineralization of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Defined Fungal-Bacterial Cocultures
Authors: Boonchan Sudarat , Britz Margaret L. , Stanley Grant A. 
Published: July 2002

ABSTRACT

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (
Stenotrophomonas maltophilia
VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (
Penicillium janthinellum
VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[
a
]pyrene cometabolically when pyrene was also present in BSM.
P. janthinellum
VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[
a
]anthracene, benzo[
a
]pyrene, and dibenz[
a
,
h
]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[
a
]anthracene, benzo[
a
]pyrene, and dibenz[
a
,
h
]anthracene, each as a single PAH in BSM, occurred when
P. janthinellum
VUO 10,201 and either bacterial consortium VUN 10,009 or
S. maltophilia
VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[
a
]pyrene was mineralized to CO
2
by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[
a
]pyrene mineralization (53% of added [
14
C]benzo[
a
]pyrene was recovered as
14
CO
2
in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.
Fusarium
Species from Nepalese Rice and Production of Mycotoxins and Gibberellic Acid by Selected Species
Authors: Desjardins A. E. , Manandhar H. K. , Plattner R. D. , Manandhar G. G. , Poling S. M. , Maragos C. M. 
Published: July 2002

ABSTRACT

Infection of cereal grains with
Fusarium
species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated
Fusarium
species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant
Fusarium
species in surface-disinfested seeds with husks were species of the
Gibberella fujikuroi
complex, including
G. fujikuroi
mating population A (anamorph,
Fusarium verticillioides
),
G. fujikuroi
mating population C (anamorph,
Fusarium fujikuroi
), and
G. fujikuroi
mating population D (anamorph,
Fusarium proliferatum
). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were
Gibberella zeae
(anamorph,
Fusarium graminearum
) and
Fusarium semitectum
, with
Fusarium acuminatum
,
Fusarium anguioides
,
Fusarium avenaceum
,
Fusarium chlamydosporum
,
Fusarium equiseti
, and
Fusarium oxysporum
occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of
G. zeae
produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of
G. zeae
, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with
Fusarium
mycotoxins.
Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes
Authors: Peters Sabine , Koschinsky Stefanie , Schwieger Frank , Tebbe Christoph C. 
Published: July 2002

ABSTRACTA cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions ofCandida kruseiandCandida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.
Degradation of Pectins with Different Degrees of Esterification by
Bacteroides thetaiotaomicron
Isolated from Human Gut Flora
Authors: Dongowski Gerhard , Lorenz Angelika , Anger Horst 
Published: July 2002

ABSTRACT

A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin.
Bacteroides thetaiotaomicron
was tested as a pectin-degrading microorganism alone and in coculture with
Escherichia coli
. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of
B. thetaiotaomicron
were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of
B. thetaiotaomicron
and
E. coli
exhibited intermediate levels of degradation activity. In pure cultures of
E. coli
no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and
B. thetaiotaomicron
was accompanied by increased formation of short-chain fatty acids.
Effect of Three Factors in Cheese Production (pH, Salt, and Heat) onMycobacterium aviumsubsp.paratuberculosisViability
Authors: Sung Nackmoon , Collins Michael T. 
Published: July 2002

ABSTRACTLow pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation forMycobacterium aviumsubsp.paratuberculosisstrains ATCC 19698 and Dominic were measured at 20°C under different pH and NaCl conditions commonly used in processing cheese. The correspondingDvalues (decimal reduction times; the time required to kill 1 log10concentration of bacteria) were measured. Also measured were theDvalues for heat-treated and nonheatedM. aviumsubsp.paratuberculosisin 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration ofM. aviumsubsp.paratuberculosis. NaCl had little or no effect on the inactivation ofM. aviumsubsp.paratuberculosis, and increasing NaCl concentrations were not associated with decreasingDvalues (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasingDvalues ofM. aviumsubsp.paratuberculosisin the acetate buffer. TheDvalues for heat-treatedM. aviumsubsp.paratuberculosisATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treatedM. aviumsubsp.paratuberculosisstrains had lowerDvalues than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. TheDvalue for heat-treatedM. aviumsubsp.paratuberculosisATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 103cells ofM. aviumsubsp.paratuberculosisper ml.
Detection of Infectious
Cryptosporidium parvum
Oocysts in Mussels (
Mytilus galloprovincialis
) and Cockles (
Cerastoderma edule
)
Authors: Gomez-Bautista M. , Ortega-Mora L. M. , Tabares E. , Lopez-Rodas V. , Costas E. 
Published: July 2002

ABSTRACT

Infective
Cryptosporidium parvum
oocysts were detected in mussels (
Mytilus galloprovincialis
) and cockles (
Cerastoderma edule
) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring
C. parvum
oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10
3
oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of
C. parvum
infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.
Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds
Authors: Xu Feng , Kulys Juozas J. , Duke Kyle , Li Kaichang , Krikstopaitis Kastis , Deussen Heinz-Josef W. , Abbate Eric , Galinyte Vilija , Schneider Palle 
Published: July 2002

ABSTRACT1-Hydroxybenzotriazole, violuric acid, andN-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.
Altered Regulation of 15-Acetyldeoxynivalenol Production in
Fusarium graminearum
Authors: Chen Lifeng , McCormick Susan P. , Hohn Thomas M. 
Published: July 2002

ABSTRACT

Most
Fusarium graminearum
isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed
F. graminearum
with both a reporter gene containing a homologous trichothecene pathway gene promoter (
TRI5
) and a gene encoding a heterologous trichothecene pathway transcription factor (
TRI6
). The
TRI5
and
TRI6
genes are part of the trichothecene pathway gene clusters of both
Fusarium sporotrichioides
and
F. graminearum
. These genes encode trichodiene synthase (encoded by
TRI5
), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by
TRI6
) required for pathway gene expression. Transformation of
F. graminearum
with plasmids containing either an
F. graminearum TRI5
promoter fragment (

FGTRI5
P

) or

FGTRI5
P

coupled with the β-
d
-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the

FGTRI5
P

sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the
F. sporotrichioides TRI6
gene (
FSTRI16
) into
F. graminearum
. The effects of
FSTRI6
on 15-ADON production also depended on plasmid integration via homologous recombination of the

FGTRI5
P

fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration to

FGTRI5
P

alters pathway gene expression and are consistent with the proposed conservation of TRI6 function between
Fusarium
species.
Gene Cloning and Expression and Secretion of
Listeria monocytogenes
Bacteriophage-Lytic Enzymes in
Lactococcus lactis
Authors: Gaeng Susanne , Scherer Siegfried , Neve Horst , Loessner Martin J. 
Published: July 2002

ABSTRACT

Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen
Listeria monocytogenes
encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in
Listeria
peptidoglycan. Ply118 is a 30.8-kDa
l
-alanoyl-
d
-glutamate peptidase and Ply511 (36.5 kDa) acts as
N
-acetylmuramoyl-
l
-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against
L. monocytogenes
contaminations, we have introduced
ply118
and
ply511
into
Lactococcus lactis
MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore,
ply511
was genetically fused with the
SP
slpA
nucleotide sequence encoding the
Lactobacillus brevis
S-layer protein signal peptide. Expression of
SP
slpA–ply511
from pSL-PL511 resulted in secretion of functional Ply511 enzyme from
L. lactis
cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3′-end coding sequence of
SP
slpA–ply511
, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Δ(S262–K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent
SP
SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of
L. monocytogenes
cells. Moreover, transformation of pSL-PL511ΔC into
L. lactis
Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.
Screening of Environmental DNA Libraries for the Presence of Genes Conferring Lipolytic Activity on
Escherichia coli
Authors: Henne Anke , Schmitz Ruth A. , Bömeke Mechthild , Gottschalk Gerhard , Daniel Rolf 
Published: July 2002

ABSTRACT

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on
Escherichia coli
clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000
E. coli
clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.