bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria
Authors: Sybesma Wilbert , Starrenburg Marjo , Tijsseling Linda , Hoefnagel Marcel H. N. , Hugenholtz Jeroen
Published: August 2003

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly.
Lactococcus lactis
,
Streptococcus thermophilus
, and
Leuconostoc
spp. all produced folate, while most
Lactobacillus
spp., with the exception of
Lactobacillus plantarum
, were not able to produce folate. Folate production was further investigated in
L
.
lactis
as a model organism for metabolic engineering and in
S
.
thermophilus
for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production.
S
.
thermophilus
excreted more folate than
L
.
lactis
, presumably as a result of differences in the number of glutamyl residues of the folate produced. In
S
.
thermophilus
5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in
L
.
lactis
5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in
S
.
thermophilus
, but pH had no effect on folate excretion by
L
.
lactis
. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of
p
-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.
Ubiquity and Persistence of
Escherichia coli
in a Midwestern Coastal Stream
Authors: Byappanahalli Muruleedhara , Fowler Melanie , Shively Dawn , Whitman Richard
Published: August 2003

Dunes Creek, a small Lake Michigan coastal stream that drains sandy aquifers and wetlands of Indiana Dunes, has chronically elevated
Escherichia coli
levels along the bathing beach near its outfall. This study sought to understand the sources of
E. coli
in Dunes Creek's central branch. A systematic survey of random and fixed sampling points of water and sediment was conducted over 3 years.
E. coli
concentrations in Dunes Creek and beach water were significantly correlated. Weekly monitoring at 14 stations during 1999 and 2000 indicated chronic loading of
E. coli
throughout the stream. Significant correlations between
E. coli
numbers in stream water and stream sediment, submerged sediment and margin, and margin and 1 m from shore were found. Median
E. coli
counts were highest in stream sediments, followed by bank sediments, sediments along spring margins, stream water, and isolated pools; in forest soils,
E. coli
counts were more variable and relatively lower. Sediment moisture was significantly correlated with
E. coli
counts. Direct fecal input inadequately explains the widespread and consistent occurrence of
E. coli
in the Dunes Creek watershed; long-term survival or multiplication or both seem likely. The authors conclude that (i)
E. coli
is ubiquitous and persistent throughout the Dunes Creek basin, (ii)
E. coli
occurrence and distribution in riparian sediments help account for the continuous loading of the bacteria in Dunes Creek, and (iii) ditching of the stream, increased drainage, and subsequent loss of wetlands may account for the chronically high
E. coli
levels observed.
Real-Time PCR for Simultaneous Detection and Quantification of
Borrelia burgdorferi
in Field-Collected
Ixodes scapularis
Ticks from the Northeastern United States
Authors: Wang Guiqing , Liveris Dionysios , Brei Brandon , Wu Hongyan , Falco Richard C. , Fish Durland , Schwartz Ira
Published: August 2003

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the
Borrelia burgdorferi
-specific
recA
gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete,
B
.
burgdorferi
, in field-collected
Ixodes scapularis
ticks. The sensitivity of qPCR for detection of
B
.
burgdorferi
DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498
I
.
scapularis
ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (
P
= 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of
B
.
burgdorferi
(
P
= 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of
k
= 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of
B
.
burgdorferi
infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
Rapid Identification ofCandidaSpecies by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy
Authors: Himmelreich Uwe , Somorjai Ray L. , Dolenko Brion , Lee Ok Cha , Daniel Heide-Marie , Murray Ronan , Mountford Carolyn E. , Sorrell Tania C.
Published: August 2003

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genusCandidato characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates ofCandida albicans,C. glabrata,C. krusei,C. parapsilosis, andC. tropicalisresulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates ofC. parapsilosisandC. glabratawith unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.
Engineering
Deinococcus geothermalis
for Bioremediation of High-Temperature Radioactive Waste Environments
Authors: Brim Hassan , Venkateswaran Amudhan , Kostandarithes Heather M. , Fredrickson James K. , Daly Michael J.
Published: August 2003

Deinococcus geothermalis
is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile
Deinococcus radiodurans
, which is being engineered for in situ bioremediation of radioactive wastes. We report that
D
.
geothermalis
is transformable with plasmids designed for
D
.
radiodurans
and have generated a Hg(II)-resistant
D
.
geothermalis
strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally,
D
.
geothermalis
is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.
Microbial Quality and Direct PCR Identification of Lactic Acid Bacteria and Nonpathogenic Staphylococci from Artisanal Low-Acid Sausages
Authors: Aymerich T. , Martín B. , Garriga M. , Hugas M.
Published: August 2003

Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment,Lactobacillus sakeiandLactobacillus curvatuswere detected in 11.8% of the samples, andLactobacillus plantarumandStaphylococcus xylosuswere detected in 17.6%. Enriched samples allowed the detection ofL. sakeiandS. xylosusin all of the samples (100%) and ofEnterococcus faeciumin 11.8% of the sausages. The percentages ofL. curvatus,L. plantarum,Staphylococcus carnosus, andStaphylococcus epidermidisvaried depending on the sausage type.L. curvatuswas detected in 80% of fuets and in 57% of chorizos.L. plantarumwas found in 50% of fuets and 100% of chorizos.S. epidermidiswas detected in only 11.8% of fuets, andS. carnosuswas detected in only 5.9% of chorizos.Lactococcus lactis,Staphylococcus warneri, andStaphylococcus simulanswere not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts ofEnterobacteriaceaeand did not contain any of the food-borne pathogens assayed.
A Newly Discovered
Bacteroides
Conjugative Transposon, CTnGERM1, Contains Genes Also Found in Gram-Positive Bacteria
Authors: Wang Yanping , Wang Gui-Rong , Shelby Aikiesha , Shoemaker Nadja B. , Salyers Abigail A.
Published: August 2003

Results of a recent study of antibiotic resistance genes in human colonic
Bacteroides
strains suggested that gene transfer events between members of this genus are fairly common. The identification of
Bacteroides
isolates that carried an erythromycin resistance gene,
ermG
, whose DNA sequence was 99% identical to that of an
ermG
gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into
Bacteroides
species from other genera. Six of seven
ermG-
containing
Bacteroides
strains tested were able to transfer
ermG
by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying
ermG
in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in
Bacteroides
isolates—CTns of the CTnERL/CTnDOT family—which sometimes carry another type of
erm
gene,
ermF
. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the
ermG
-containing strains but also to DNA from strains that did not carry
ermG
. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired
ermG
. The percentage of G+C content of the
ermG
region was significantly lower than that of the chromosome of
Bacteroides
species—an indication that CTnGERM1 may have entered
Bacteroides
strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered
Bacteroides
species relatively recently. One of the genes located upstream of e
rmG
encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from
Streptococcus pyogenes
. Our having found >90% sequence identity of two upstream genes, including
mefA
, and the remnants of two transposon-carried genes downstream of
ermG
with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.
Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding ofCryptosporidium parvumin Adult Beef Cattle
Authors: Atwill E. R. , Hoar B. , das Graças Cabral Pereira M. , Tate K. W. , Rulofson F. , Nader G.
Published: August 2003

Our primary goal was to generate an accurate estimate of the daily environmental loading rate ofCryptosporidium parvumoocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding ofC.parvumoocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces−1, with a 54% probability of detecting just one oocyst g of feces−1. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive forC.parvumwas 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces−1. The estimated environmental loading rate ofC.parvumranged from 3,900 to 9,200 oocysts cow−1day−1, which is substantially less than a previous estimate of 1.7 × 105oocysts cow−1day−1(range of 7.7 × 104to 2.3 × 105oocysts cow−1day−1) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol.2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.
Curvularia
Haloperoxidase: Antimicrobial Activity and Potential Application as a Surface Disinfectant
Authors: Hansen Eva H. , Albertsen Line , Schäfer Thomas , Johansen Charlotte , Frisvad Jens C. , Molin Søren , Gram Lone
Published: August 2003

A presumed antimicrobial enzyme system, the
Curvularia
haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide,
Curvularia
haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The
Curvularia
haloperoxidase system caused several-log-unit reductions in counts of bacteria (
Pseudomonas
spp.,
Escherichia coli
,
Serratia marcescens
,
Aeromonas salmonicida
,
Shewanella putrefaciens
,
Staphylococcus epidermidis
, and
Listeria monocytogenes
), yeasts (
Candida
sp. and
Rhodotorula
sp.), and filamentous fungi (
Aspergillus niger
,
Aspergillus tubigensis
,
Aspergillus versicolor
,
Fusarium oxysporum
,
Penicillium chrysogenum
, and
Penicillium paxilli
) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of
S. marcescens
and
S. epidermidis
cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the
Curvularia
haloperoxidase system. The killing effect of the
Curvularia
haloperoxidase system was rapid, and 10
6
CFU of
E. coli
cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against
E. coli
10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the
Curvularia
haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the
Curvularia
haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.
Characterization of the Corrinoid Iron-Sulfur ProteinTetrachloroethene Reductive Dehalogenase of
Dehalobacterrestrictus
Authors: Maillard Julien , Schumacher Wolfram , Vazquez Francisco , Regeard Christophe , Hagen Wilfred R. , Holliger Christof
Published: August 2003

The
membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase)
(PceA; EC 1.97.1.8), the terminal component of the respiratory chain of
Dehalobacter restrictus
, was purified 25-fold to apparent
electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis revealed a single band with an apparent molecular mass
of 60 ± 1 kDa, whereas the native molecular mass was 71±
8 kDa according to size exclusion chromatography in the
presence of the detergent octyl-β-
d
-glucopyranoside.
The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0±
0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1±
0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile
sulfur. Purified PceA catalyzed the reductive dechlorination of
tetrachloroethene and trichloroethene to
cis
-1,2-dichloroethene with a specific activity of
250 ± 12 nkat/mg of protein. In addition, several
chloroethanes and tetrachloromethane caused methyl viologen oxidation
in the presence of PceA. The

K
m

values for
tetrachloroethene, trichloroethene, and methyl viologen were 20.4±
3.2, 23.7 ± 5.2, and 47 ± 10 μM,
respectively. The PceA exhibited the highest activity at pH 8.1 and was
oxygen sensitive, with a half-life of activity of 280 min upon exposure
to air. Based on the almost identical N-terminal amino acid sequences
of PceA of
Dehalobacter restrictus
,
Desulfitobacterium
hafniense
strain TCE1 (formerly
Desulfitobacterium
frappieri
strain TCE1), and
Desulfitobacterium hafniense
strain PCE-S (formerly
Desulfitobacterium frappieri
strain
PCE-S), the
pceA
genes of the first two organisms were cloned
and sequenced. Together with the
pceA
genes of
Desulfitobacterium hafniense
strains PCE-S and Y51, the
pceA
genes of
Desulfitobacterium hafniense
strain TCE1 and
Dehalobacter restrictus
form a coherent group
of reductive dehalogenases with almost 100% sequence identity.
Also, the
pceB
genes, which may code for a membrane anchor
protein of PceA, and the intergenic regions of
Dehalobacter
restrictus
and the three desulfitobacteria had identical
sequences. Whereas the
cprB
(chlorophenol reductive
dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always
located upstream of
cprA
, all
pceB
genes known so far
are located downstream of
pceA.
The possible consequences of
this feature for the annotation of putative reductive dehalogenase
genes are discussed, as are the sequence around the iron-sulfur cluster
binding motifs and the type of iron-sulfur clusters of the reductive
dehalogenases of
Dehalobacter restrictus
and
Desulfitobacterium dehalogenans
identified by electron
paramagnetic resonance
spectroscopy.
Genetic Diversity and Temporal Variation in the Cyanophage Community Infecting Marine
Synechococcus
Species in Rhode Island's Coastal Waters
Authors: Marston Marcia F. , Sallee Jennifer L.
Published: August 2003

The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10
4
phage ml
−1
in the summer months to less then 10
2
phage ml
−1
during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of
Synechococcus
spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g.,
Podoviridae
or
Siphoviridae
) may be important components of the Rhode Island cyanophage community.
Effect of Incubation Temperature on Isolation of
Campylobacter jejuni
Genotypes from Foodstuffs Enriched in Preston Broth
Authors: Scates Pam , Moran Lynn , Madden Robert H.
Published: August 2003

Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (
n
= 24) and raw lamb liver (
n
= 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two
Campylobacter
isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all
Campylobacter jejuni
, as were 96% of the poultry isolates, with the remainder being
Campylobacter lari
. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the
C. jejuni
isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD
C. jejuni
genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of
C. jejuni
, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.
PCR-Restriction Fragment Length Polymorphism Method for Detection of
Cyclospora cayetanensis
in Environmental Waters without Microscopic Confirmation
Authors: Shields Joan M. , Olson Betty H.
Published: August 2003

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of
Cyclospora cayetanensis
in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme
Alu
I, differentiates the amplified target sequence from
C
.
cayetanensis
from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with
Mnl
I (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other
Cyclospora
species, they were not
C
.
cayetanensis
. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.
virF-PositiveYersinia pseudotuberculosisandYersinia enterocoliticaFound in Migratory Birds in Sweden
Authors: Niskanen Taina , Waldenström Jonas , Fredriksson-Ahomaa Maria , Olsen Björn , Korkeala Hannu
Published: August 2003

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total,Yersiniaspp. were isolated from 12.8% of collected samples. The most commonly found species wasYersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed byY. intermedia(3.8%),Y. frederiksenii(3.0%),Y. kristensenii(0.9%),Y. pseudotuberculosis(0.6%), andY. rohdei(0.4%). The pathogenic,virF-positiveY. pseudotuberculosisstrains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis withNotI andSpeI enzymes. In addition, 10Y. enterocoliticastrains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying thevirFgene on their plasmids. All pathogenicY. pseudotuberculosisandY. enterocoliticastrains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.
Alternative Splicing of Transcripts from
crtI
and
crtYB
Genes of
Xanthophyllomyces dendrorhous
Authors: Lodato P. , Alcaino J. , Barahona S. , Retamales P. , Cifuentes V.
Published: August 2003

Xanthophyllomyces dendrorhous
is one of the relevant sources of the carotenoid astaxanthin. In this paper, we describe for the first time cloning of unexpected cDNAs obtained from the
crtI
and
crtYB
genes of
X. dendrorhous
strain UCD 67-385. The cDNA of the
crtI
gene conserves 80 bp of the first intron, while the cDNA of the
crtYB
gene conserves 55 bp of the first intron and lacks 111 bp of the second exon. The
crtI
and
crtYB
RNAs could be spliced in alternative splice sites, which produced alternative transcripts which could not be translated to active CRTI and CRTYB proteins since they had numerous stop codons in their sequences. The ratio of mature mRNA to alternative mRNA for the
crtI
gene decreased as a function of the age of the culture, while the cellular content of carotenoids increased. It is possible that splicing to mature or alternative transcripts could regulate the cellular concentrations of phytoene desaturase and phytoene synthase-lycopene cyclase proteins, depending on the physiological or environmental conditions.
Growth of
Rhodosporidium toruloides
Strain DBVPG 6662 on Dibenzothiophene Crystals and Orimulsion
Authors: Baldi Franco , Pepi Milva , Fava Fabio
Published: August 2003

Strains DBVPG 6662 and DBVPG 6739 of
Rhodosporidium toruloides
, a basidiomycete yeast, grew on thiosulfate as a sulfur source and glucose (2 g liter
−1
or 10.75 mM) as a carbon source. DBVPG 6662 has a defective sulfate transport system, whereas DBVPG 6739 barely grew on sulfate. They were compared for the ability to use dibenzothiophene (DBT) and related organic sulfur compounds as sulfur sources. In the presence of glucose as a carbon source and DBT as a sulfur source, strain DBVPG 6662 grew better than DBVPG 6739. In the presence of thiosulfate as a sulfur source, the two yeast strains did not use DBT, DBT-sulfone, benzenesulfonic acid, biphenyl, and fluorene. When the two strains were grown in the presence of glucose, strain DBVPG 6662 transformed 27% of the DBT present (10 μM) at a rate of 0.023 μmol liter
−1
h
−1
in 36 h. Traces of 2,2′-dihydroxylated biphenyl were transiently accumulated under these conditions. When the same strain was grown on glucose in the presence of a higher concentration of DBT (0.5 g liter
−1
), mainly in an insoluble form, the whole surface of the DBT crystals was colonized by a thick mycelium. This adherent structure was imaged by confocal microscopy with fluorescent concanavalin A, a lectin that specifically binds glucose and mannose residues. When DBVPG 6662 was grown on glucose in the presence of a commercial emulsion of bitumen, i.e., orimulsion, 68% of the benzo- and dibenzothiophenes and DBTs was removed after 15 days of incubation. The fungus adhered by hyphae to orimulsion droplets. When cultivated in the presence of commercial emulsifier-free fuel oil containing alkylated benzothiophenes and DBTs and having a composition similar to that of orimulsion, strain DBVPG 6662 removed only 11% of the total organic sulfur that occurs in the medium and did not adhere to the oil droplets. These results indicate that strain DBVPG 6662 is able to utilize the organic sulfur of DBT and a large variety of thiophenic compounds that occur extensively in commercial fuel oils by physically adhering to the organic sulfur source.
Rapid Detection of Meat Spoilage by Measuring Volatile Organic Compounds by Using Proton Transfer Reaction Mass Spectrometry
Authors: Mayr D. , Margesin R. , Klingsbichel E. , Hartungen E. , Jenewein D. , Schinner F. , Märk T. D.
Published: August 2003

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4°C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria,
Pseudomonas
spp.,
Enterobacteriaceae
, lactic acid bacteria, and
Enterococcus
spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (
P
< 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.
Photobactin: a Catechol Siderophore Produced by
Photorhabdus luminescens
, an Entomopathogen Mutually Associated with
Heterorhabditis bacteriophora
NC1 Nematodes
Authors: Ciche Todd A. , Blackburn Michael , Carney John R. , Ensign Jerald C.
Published: August 2003

The nematode
Heterorhabditis bacteriophora
transmits a monoculture of
Photorhabdus luminescens
bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an
ngrA
mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and
ngrA
is homologous to a siderophore biosynthetic gene,
entD
. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn
5
-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn
5
transposon was inserted into
phbH
; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of
Vibrio cholerae
. Other putative siderophore biosynthetic and transport genes flanking
phbH
were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the
ngrA
mutant in supporting nematode growth and reproduction.
Occurrence of
Escherichia coli
and Enterococci in
Cladophora
(Chlorophyta) in Nearshore Water and Beach Sand of Lake Michigan
Authors: Whitman Richard L. , Shively Dawn A. , Pawlik Heather , Nevers Meredith B. , Byappanahalli Muruleedhara N.
Published: August 2003

Each summer, the nuisance green alga
Cladophora
(mostly
Cladophora glomerata
) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment. Traditionally, elevated counts of
Escherichia coli
are presumed to indicate the presence of sewage, mostly derived from nearby point sources. The relationship between fecal indicator bacteria and
Cladophora
remains essentially unstudied. This investigation describes the local and regional density of
Escherichia coli
and enterococci in
Cladophora
mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan. Samples of
Cladophora
strands collected from 10 beaches (
n
= 41) were assayed for concentrations of
E. coli
and enterococci during the summer of 2002. Both
E. coli
and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (± standard errors) of 5.3 (± 4.8) and 4.8 (± 4.5) per g (dry weight).
E. coli
and enterococci were strongly correlated in southern Lake Michigan beaches (
P
< 0.001,
R
2
= 0.73,
n
= 17) but not in northern beaches (
P
= 0.892,
n
= 16). Both
E. coli
and enterococci survived for over 6 months in sun-dried
Cladophora
mats stored at 4°C; the residual bacteria in the dried alga readily grew upon rehydration. These findings suggest that
Cladophora
amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of
E. coli
and enterococci as indicators of water quality for freshwater recreational beaches.
Intraspecies Polymorphism of
Cryptosporidium parvum
Revealed by PCR-Restriction Fragment Length Polymorphism (RFLP) and RFLP-Single-Strand Conformational Polymorphism Analyses
Authors: Wu Zhiliang , Nagano Isao , Boonmars Thidarut , Nakada Takumi , Takahashi Yuzo
Published: August 2003

A glycoprotein (Cpgp40/15)-encoding gene of
Cryptosporidium parvum
was analyzed to reveal intraspecies polymorphism within
C
.
parvum
isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of
C
.
parvum
.
Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting
Saccharomyces cerevisiae
Improves Ethanol Production
Authors: Roca Christophe , Nielsen Jens , Olsson Lisbeth
Published: August 2003

Cofactor imbalance impedes xylose assimilation in
Saccharomyces cerevisiae
that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant
S. cerevisiae
by deleting
GDH1
, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either
GDH2
, which encodes an NADH-dependent glutamate dehydrogenase, or
GLT1
and
GLN1
, which encode the GS-GOGAT complex. Overexpression of
GDH2
increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol
−1
, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.
Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in
Escherichia coli
by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling
Authors: Choi Jong Hyun , Lee Sang Jun , Lee Seok Jae , Lee Sang Yup
Published: August 2003

The transcriptome profiles of recombinant
Escherichia coli
producing human insulin-like growth factor I fusion protein (IGF-I
f
) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-I
f
after induction. Among these down-regulated genes, we rationally selected and coexpressed in
E
.
coli
producing IGF-I
f
the
prsA
gene (encoding a phosphoribosyl pyrophosphate synthetase) and the
glpF
gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I
f
could be increased from 1.8 ± 0.13 (± standard deviation) to 4.3 ± 0.24 g/liter. The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.
An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing
Lactobacillus fermentum
Strain, and Determination of Its Cholesterol-Lowering Properties
Authors: Pereira Dora I. A. , McCartney Anne L. , Gibson Glenn R.
Published: August 2003

This study evaluated the use of a bile-salt-hydrolyzing
Lactobacillus fermentum
strain as a probiotic with potential hypocholesterolemic properties. The effect of
L. fermentum
on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the
Lactobacillus
strain was evaluated. Administration of
L. fermentum
resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial,
Lactobacillus
,
Bacteroides
, and clostridial populations through
L. fermentum
supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the
L. fermentum
strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that
L. fermentum
KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.
Characterization of
Streptococcus mutans
Strains Deficient in EIIAB
Man
of the Sugar Phosphotransferase System
Authors: Abranches Jacqueline , Chen Yi-Ywan M. , Burne Robert A.
Published: August 2003

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB
Man
(encoded by
manL
) in gene regulation and sugar transport was investigated in
Streptococcus mutans
UA159. The
manL
knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB
Man
is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (
fruI
) and constitutive (
fruCD
) EII fructose, revealed that
S. mutans
EIIAB
Man
transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB
Man
controlled expression of the known virulence genes, glucosyltransferases (
gtfBC
) and fructosyltransferase (
ftf
) promoter fusions of these genes were established in the wild-type and EIIAB
Man
-deficient strains. In the
manL
mutant, the level of chloramphenicol acetyltransferase activity expressed from the
gtfBC
promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB
Man
is required for optimal expression of
gtfBC
. No significant differences were observed between the mutant and the wild-type background in
ftf
regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in
ftf
expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB
Man
-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB
Man
has a pleiotropic effect on gene expression.
Effects of a Chlorhexidine Gluconate-Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems
Authors: McBain Andrew J. , Bartolo Robert G. , Catrenich Carl E. , Charbonneau Duane , Ledder Ruth G. , Gilbert Peter
Published: August 2003

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (
P
< 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in
Prevotella
sp. and
Selenomonas infelix
. Pure-culture studies of 10 oral bacteria (eight genera) showed that
Actinomyces naeslundii
,
Veillonella dispar
,
Prevotella nigrescens
, and the streptococci were highly susceptible to CHX, while
Lactobacillus rhamnosus
,
Fusobacterium nucleatum
, and
Neisseria subflava
were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (
P
< 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.
Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation
Authors: Higgins Vincent J. , Beckhouse Anthony G. , Oliver Anthony D. , Rogers Peter J. , Dawes Ian W.
Published: August 2003

Genome-wide expression analysis of an industrial strain ofSaccharomyces cerevisiaeduring the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of theERGgenes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similarERG10andTRR1gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed thatergmutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.
Detection and Quantification ofPlectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting
Authors: Atkins S. D. , Clark I. M. , Sosnowska D. , Hirsch P. R. , Kerry B. R.
Published: August 2003

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungusPlectosphaerella cucumerinahas demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitorP. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence ofP. cucumerinain soil, root, and nematode samples. PCR was combined with a bait method to identifyP. cucumerinafrom infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A secondP.cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance ofP. cucumerinafrom two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity ofP. cucumerinato be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.