bluuebunny/crossref_metadata_2025 huggingface dataset:
Penetration of Rifampin through
Staphylococcus epidermidis
Biofilms
Authors: Zheng Zhilan , Stewart Philip S.
Published: July 2002

Rifampin penetrated biofilms formed by
Staphylococcus epidermidis
but failed to effectively kill the bacteria. Penetration was demonstrated by a simple diffusion cell bioassay and by transmission electron microscopic observation of antibiotic-affected cells at the distal edge of the biofilm.
In Vitro Interactions of Artemisinin with Atovaquone, Quinine, and Mefloquine againstPlasmodium falciparum
Authors: Gupta S. , Thapar M. M. , Wernsdorfer W. H. , Björkman A.
Published: July 2002

The interactions of artemisinin with atovaquone, quinine, and mefloquine were investigated in threePlasmodium falciparumstrains (strains F-32, FCR-3, and K-1) by an in vitro culture assay. The parasites were cultured for 48 h in the presence of different concentrations and proportions of two drugs at a time in a checkerboard design. The response parameters were determined, and the sums of the fractional inhibitory concentrations (ΣFICs) of the drug combinations were calculated for different degrees of inhibition (50% effective concentration [EC50], EC90, and EC99). Within therapeutically relevant molar ratios (19 to 200), the combination of quinine and artemisinin showed mean ΣFICs of 1.71 at the EC50, 0.36 at the EC90, and 0.13 at the EC99, indicating increasing synergism. Within the range of molar ratios of 4.3 to 50, the combination of mefloquine and artemisinin yielded mean ΣFCIs of 0.93, 0.44, and 0.31 at the EC50, EC90, and EC99, respectively, indicating synergism. The atovaquone combination showed additive activity to synergism at atovaquone/artemisinin proportions considered relevant to the in vivo situation, i.e., between 4.3 and 200, with the mean ΣFICs decreasing from 1.34 at the EC50to 0.85 and 0.23 at the EC90and EC99, respectively. Interstrain differences in the degree of drug interaction were seen with the three strains for all combinations. Synergism was most consistent with quinine.
Shiga-Like Toxin II Derived fromEscherichia coliO157:H7 Modifies Renal Handling of Levofloxacin in Rats
Authors: Zhao Ying Lan , Cen Xiao Bo , Ito Masafumi , Yokoyama Keiko , Takagi Kenji , Kitaichi Kiyoyuki , Nadai Masayuki , Ohta Michio , Takagi Kenzo , Hasegawa Takaaki
Published: July 2002

The effect of Shiga-like toxin II (SLT-II) (2 μg/animal), which was derived fromEscherichia coliO157:H7, on renal handling of levofloxacin (LVX), a model drug for quinolone antimicrobial agents, was investigated in rats 24 h after intravenous injection. In histopathological examination, acute tubular injury was observed in SLT-II-treated rats, but the glomeruli were not injured. SLT-II significantly increased the steady-state concentration of LVX in plasma to 1.5-fold that of control rats. SLT-II induced significant decreases in the glomerular filtration rate (GFR) and renal clearance (CLR) of LVX. SLT-II slightly, but significantly, increased the unbound fraction and decreased renal plasma flow with no change in the extraction ratio ofp-aminohippurate. SLT-II significantly increased concentrations of tumor necrosis factor alpha (TNF-α) and nitrite and nitrate (NOx) in plasma. The TNF-α inhibitor pentoxifylline partly, but significantly, inhibited SLT-II-induced decreases in the GFR and CLRof LVX; in contrast,S-methylisothiourea, a selective inhibitor of inducible nitric oxide synthase, did not. Western blotting analysis revealed that SLT-II did not alter the levels of multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein in kidneys 24 h after injection, assuming the lack of involvement of Mrp2 and P-glycoprotein in SLT-II-induced acute renal tubular injury and renal handling of LVX observed 24 h after SLT-II injection. The present study suggests that SLT-II impairs the renal handling of LVX by decreasing GFR and causing decreased renal plasma flow.
Mechanism of Action ofMelaleuca alternifolia(Tea Tree) Oil onStaphylococcus aureusDetermined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy
Authors: Carson Christine F. , Mee Brian J. , Riley Thomas V.
Published: July 2002

The essential oil ofMelaleuca alternifolia(tea tree) has broad-spectrum antimicrobial activity. The mechanisms of action of tea tree oil and three of its components, 1,8-cineole, terpinen-4-ol, and α-terpineol, againstStaphylococcus aureusATCC 9144 were investigated. Treatment with these agents at their MICs and two times their MICs, particularly treatment with terpinen-4-ol and α-terpineol, reduced the viability ofS. aureus. None of the agents caused lysis, as determined by measurement of the optical density at 620 nm, although cells became disproportionately sensitive to subsequent autolysis. Loss of 260-nm-absorbing material occurred after treatment with concentrations equivalent to the MIC, particularly after treatment with 1,8-cineole and α-terpineol.S. aureusorganisms treated with tea tree oil or its components at the MIC or two times the MIC showed a significant loss of tolerance to NaCl. When the agents were tested at one-half the MIC, only 1,8-cineole significantly reduced the tolerance ofS. aureusto NaCl. Electron microscopy of terpinen-4-ol-treated cells showed the formation of mesosomes and the loss of cytoplasmic contents. The predisposition to lysis, the loss of 260-nm-absorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane.
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

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

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

A 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

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

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

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

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

A 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

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

Low 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

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

1-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

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

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

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.
Decolorization and Detoxification of Textile Dyes with a Laccase from
Trametes hirsuta
Authors: Abadulla Elias , Tzanov Tzanko , Costa Silgia , Robra Karl-Heinz , Cavaco-Paulo Artur , Gübitz Georg M.
Published: July 2002

Trametes hirsuta
and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the
T. hirsuta
laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC
50
) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of
Pseudomonas putida
) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with
T. hirsuta
or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes.
Role of ςBin Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation inListeria monocytogenes
Authors: Ferreira Adriana , O'Byrne Conor P. , Boor Kathryn J.
Published: July 2002

To determine the contribution of sigma B (ςB) to survival of stationary-phaseListeria monocytogenescells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolarsigBnull mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigBand wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigBstrain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigBstrain. These results suggest the existence inL. monocytogenesof both a ςB-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ςBcontributed to resistance to both oxidative stress and carbon starvation inL. monocytogenes. The ΔsigBstrain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigBstrain lost viability more rapidly than the parent strain. ςBcontributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ςBplays a role in protectingL. monocytogenesagainst environmental adversities.
Determination of Pyrimidine Dimers in
Escherichia coli
and
Cryptosporidium parvum
during UV Light Inactivation, Photoreactivation, and Dark Repair
Authors: Oguma Kumiko , Katayama Hiroyuki , Mitani Hiroshi , Morita Shigemitsu , Hirata Tsuyoshi , Ohgaki Shinichiro
Published: July 2002

UV inactivation, photoreactivation, and dark repair of
Escherichia coli
and
Cryptosporidium parvum
were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of
E. coli
, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of
E. coli
. The colony-forming ability of
E. coli
also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When
E. coli
were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however,
E. coli
showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When
C. parvum
were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation,
C. parvum
also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of
C. parvum
would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.
Microbial Communities in the Chemocline of a Hypersaline Deep-Sea Basin (Urania Basin, Mediterranean Sea)
Authors: Sass Andrea M. , Sass Henrik , Coolen Marco J. L. , Cypionka Heribert , Overmann Jörg
Published: July 2002

The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of ∼3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria,
Alteromonas macleodii
, and
Halomonas aquamarina
. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unsually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.
Prevalence ofCampylobacterspp., Escherichia coli, andSalmonellaSerovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area
Authors: Zhao Cuiwei , Ge Beilei , De Villena Juan , Sudler Robert , Yeh Emily , Zhao Shaohua , White David G. , Wagner David , Meng Jianghong
Published: July 2002

A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence ofEscherichia coliandSalmonellaserovars, and 719 of these samples were also tested forCampylobacterspp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n= 184) were contaminated withCampylobacter, and a large percentage of the stores visited (91%) hadCampylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yieldedCampylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722Campylobacterisolates were obtained from 159 meat samples; 53.6% of these isolates wereCampylobacter jejuni, 41.3% wereCampylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yieldedE. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive forE. coli. However, only 25 (3.0%) of the retail meat samples tested were positive forSalmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.
Sequence Analysis of a 101-Kilobase Plasmid Required for Agar Degradation by aMicroscillaIsolate
Authors: Zhong Zhenping , Toukdarian Aresa , Helinski Donald , Knauf Vic , Sykes Sean , Wilkinson Jane E. , O'Bryne Colleen , Shea Terry , DeLoughery Craig , Caspi Ron
Published: July 2002

An agar-degrading marine bacterium identified as aMicroscillaspecies was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.
Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and HumanListeria monocytogenesIsolates
Authors: Vela A. I. , Fernandez-Garayzabal J. F. , Vazquez J. A. , Latre M. V. , Blanco M. M. , Moreno M. A. , de la Fuente L. , Marco J. , Franco C. , Cepeda A. , Rodriguez Moure A. A. , Suarez G. , Dominguez L.
Published: July 2002

A total of 153 strains ofListeria monocytogenesisolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains ofL. monocytogenesdisplayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the SpanishL. monocytogenesisolates studied.L. monocytogenesisolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak.L. monocytogenesstrains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.
Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes
Authors: Miller William G. , Brandl Maria T. , Quiñones Beatriz , Lindow Steven E.
Published: July 2002

A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the
scrR
sucrose repressor gene and the promoterless
gfp,
lacZ,
and
inaZ
reporter genes, respectively, fused to the
scrY
promoter from
Salmonella enterica
serovar Typhimurium. Cells of
Erwinia herbicola
containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the
inaZ
reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either
lacZ
or
gfp
. The
lacZ
reporter gene was found to be more efficient than
gfp
, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly,
inaZ
was found to be more efficient than
lacZ,
requiring 30-fold fewer cells at 1.45 μM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and
gfp
-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 μM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.
Changes in Protein Synthesis and Morphology during Acid Adaptation of
Propionibacterium freudenreichii
Authors: Jan Gwénaël , Leverrier Pauline , Pichereau Vianney , Boyaval Patrick
Published: July 2002

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of
Propionibacterium freudenreichii
was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in
P. freudenreichii
.
Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes
Authors: Charcosset Jean-Yves , Chauvet Eric
Published: July 2002

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (

r
2

= 0.83 to 0.98) and highly significant (
P
≪ 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.
Influence of the Natural Microbial Flora on the Acid Tolerance Response of
Listeria monocytogenes
in a Model System of Fresh Meat Decontamination Fluids
Authors: Samelis John , Sofos John N. , Kendall Patricia A. , Smith Gary C.
Published: July 2002

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10
5
CFU/ml)
Listeria monocytogenes
were evaluated at 35°C in water (10 or 85°C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35°C rather than lower (≤15°C) temperatures to maximize the response of inoculated
L. monocytogenes
in the washings with or without competitive flora. Acid solution washings were free (<1.0 log CFU/ml) of natural flora before inoculation (day 0), and no microbial growth occurred during storage (35°C, 8 days). Inoculated
L. monocytogenes
died off (negative enrichment) in acid washings within 24 h. In nonacid (water) washings, the pathogen increased (approximately 1.0 to 2.0 log CFU/ml), irrespective of natural flora, which, when present, predominated (>8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of
L. monocytogenes
at 35°C. In filter-sterilized water washings, inoculated
L. monocytogenes
increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.