bluuebunny/crossref_metadata_2025 huggingface dataset:
Cold Shock Induction of Thermal Sensitivity in
Listeria monocytogenes
Authors: Miller Arthur J. , Bayles Darrell O. , Eblen B. Shawn
Published: July 2002

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of
Listeria monocytogenes
. In a model broth system, thermal death time at 60°C was reduced by up to 45% after
L. monocytogenes
Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The
Z
values were 8.8°C for controls and 7.7°C for cold-shocked cells. The
D
values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine
L. monocytogenes
strains that were cold shocked for 3 h exhibited
D
60
values that were reduced by 13 to 37%. The
D
-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The
D
values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the
D
values of
L. monocytogenes
Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in
L. monocytogenes
by thermal flux shows potential to become a practical and efficacious preventative control method.
Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter
Authors: Vriesema Aldwin J. M. , Brinkman René , Kok Jan , Dankert Jacob , Zaat Sebastian A. J.
Published: July 2002

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of
Streptococcus gordonii
strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the
hydA
gene from
Clostridium acetobutylicum
, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that
S. gordonii
can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.
Diversity and Distribution of DNA Sequences with Affinity to Ammonia-Oxidizing Bacteria of the β Subdivision of the Class
Proteobacteria
in the Arctic Ocean
Authors: Bano Nasreen , Hollibaugh James T.
Published: July 2002

The spatial distribution and diversity of ammonia-oxidizing bacteria of the β subdivision of the class
Proteobacteria
(hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to
Escherichia coli
16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine
Nitrosospira
-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine
Nitrosomonas
-like sequences. Our results suggest that the Arctic Ocean β-proteobacterial ammonia oxidizers have low diversity and are dominated by marine
Nitrosospira
-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.
Design and Evaluation of a
Lactobacillus manihotivorans
Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation
Authors: Ampe Frédéric
Published: July 2002

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species
Lactobacillus manihotivorans
recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the
L. manihotivorans
strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of
L. manihotivorans
in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.
Inactivation of the Glutamate Decarboxylase Gene in
Lactococcus lactis
subsp.
cremoris
Authors: Nomura Masaru , Kobayashi Miho , Ohmomo Sadahiro , Okamoto Takashi
Published: July 2002

Lactococcus lactis
subsp.
lactis
strains show glutamate decarboxylase activity, whereas
L. lactis
subsp.
cremoris
strains do not. The
gadB
gene encoding glutamate decarboxylase was detected in the
L. lactis
subsp.
cremoris
genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.
Production and Characterization of Monoclonal Antibodies against Vegetative Cells of
Bacillus cereus
Authors: Charni Nadine , Perissol Claude , Le Petit Jean , Rugani Nathalie
Published: July 2002

Two monoclonal antibodies (MAbs) against
Bacillus cereus
were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with
B. cereus
vegetative cells. They reacted with
B. cereus
vegetative cells while failing to recognize
B. cereus
spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of
B. cereus
vegetative cells in foods was investigated.
Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of
Escherichia coli
Authors: Seo Hak Soo , Koo Yeon Jong , Lim Jae Yun , Song Jong Tae , Kim Chung Ho , Kim Ju Kon , Lee Jong Seob , Choi Yang Do
Published: July 2002

To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the
Escherichia coli
genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in
E. coli
and purified to near homogeneity; its molecular weight was 88,300, as expected. The

K
m

values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the
k
cat
values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (
k
cat
/

K
m

). The

K
m

and
k
cat
values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-
d
-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.
Lon Protease Influences Antibiotic Production and UV Tolerance of
Pseudomonas fluorescens
Pf-5
Authors: Whistler Cheryl A. , Stockwell Virginia O. , Loper Joyce E.
Published: July 2002

Pseudomonas fluorescens
Pf-5 is a soil bacterium that suppresses plant pathogens due in part to its production of the antibiotic pyoluteorin. Previous characterization of Pf-5 revealed three global regulators, including the stationary-phase sigma factor ς
S
and the two-component regulators GacA and GacS, that influence both antibiotic production and stress response. In this report, we describe the serine protease Lon as a fourth global regulator influencing these phenotypes in Pf-5.
lon
mutants overproduced pyoluteorin, transcribed pyoluteorin biosynthesis genes at enhanced levels, and were more sensitive to UV exposure than Pf-5. The
lon
gene was preceded by sequences that resembled promoters recognized by the heat shock sigma factor ς
32
(ς
H
) of
Escherichia coli
, and Lon accumulation by Pf-5 increased after heat shock. Therefore, ς
H
represents the third sigma factor (with ς
S
and ς
70
) implicated in the regulation of antibiotic production by
P. fluorescens
. Lon protein levels were similar in stationary-phase and exponentially growing cultures of Pf-5 and were not positively affected by the global regulator ς
S
or GacS. The association of antibiotic production and stress response has practical implications for the success of disease suppression in the soil environment, where biological control organisms such as Pf-5 are likely to encounter environmental stresses.
Reduction of Pulmonary Toxicity of
Stachybotrys chartarum
Spores by Methanol Extraction of Mycotoxins
Authors: Rao Carol Y. , Brain Joseph D. , Burge Harriet A.
Published: July 2002

The fungus
Stachybotrys chartarum
has been implicated in cases of nonspecific indoor air quality complaints in adults and in cases of pulmonary hemorrhaging in infants. The effects that have been described have been attributed to mycotoxins. Previous dose-effect studies focused on exposure to a single mycotoxin in a solvent, a strategy which is unlikely to accurately characterize the effects of inhaled spores. In this study we examined the role of mycotoxins in the pulmonary effects caused by
S. chartarum
spores and the dose dependency of these effects.
S. chartarum
spores were extracted in methanol to reduce the mycotoxin content of the spores. Then either untreated (toxin-containing) or methanol-extracted
S. chartarum
spores were intratracheally instilled into male 10-week-old Charles River-Dawley rats. After 24 h, the lungs were lavaged, and the bronchoalveolar lavage fluid was analyzed to determine differences in lactic dehydrogenase, albumin, hemoglobin, myeloperoxidase, and leukocyte differential counts. Weight change was also monitored. Our data show that methanol extraction dramatically reduced the toxicity of
S. chartarum
spores. No statistically significant effects were observed in the bronchoalveolar lavage fluids of the animals that were treated with methanol-extracted spores at any dose. Conversely, dose-dependent effects of the toxin-containing spores were observed when we examined the lactic dehydrogenase, albumin, and hemoglobin concentrations, the polymorphonuclear leukocyte counts, and weight loss. Our findings show that a single, intense exposure to toxin-containing
S. chartarum
spores results in pulmonary inflammation and injury in a dose-dependent manner. Importantly, the effects are related to methanol-soluble toxins in the spores.
Sphingomonas alaskensis
Strain AFO1, an Abundant Oligotrophic Ultramicrobacterium from the North Pacific
Authors: Eguchi Mitsuru , Ostrowski Martin , Fegatella Fitri , Bowman John , Nichols David , Nishino Tomohiko , Cavicchioli Ricardo
Published: July 2002

Numerous studies have established the importance of picoplankton (microorganisms of ≤2 μm in length) in energy flow and nutrient cycling in marine oligotrophic environments, and significant effort has been directed at identifying and isolating heterotrophic picoplankton from the world's oceans. Using a method of diluting natural seawater to extinction followed by monthly subculturing for 12 months, a bacterium was isolated that was able to form colonies on solid medium. The strain was isolated from a 10
5
dilution of seawater where the standing bacterial count was 3.1 × 10
5
cells ml
−1
. This indicated that the isolate was representative of the most abundant bacteria at the sampling site, 1.5 km from Cape Muroto, Japan. The bacterium was characterized and found to be ultramicrosized (less than 0.1 μm
3
), and the size varied to only a small degree when the cells were starved or grown in rich media. A detailed molecular (16S rRNA sequence, DNA-DNA hybridization, G+C mol%, genome size), chemotaxonomic (lipid analysis, morphology), and physiological (resistance to hydrogen peroxide, heat, and ethanol) characterization of the bacterium revealed that it was a strain of
Sphingomonas alaskensis
. The type strain, RB2256, was previously isolated from Resurrection Bay, Alaska, and similar isolates have been obtained from the North Sea. The isolation of this species over an extended period, its high abundance at the time of sampling, and its geographical distribution indicate that it has the capacity to proliferate in ocean waters and is therefore likely to be an important contributor in terms of biomass and nutrient cycling in marine environments.
Isolation of Novel Pelagic Bacteria from the German Bight and Their Seasonal Contributions to Surface Picoplankton
Authors: Eilers Heike , Pernthaler Jakob , Peplies Jörg , Glöckner Frank Oliver , Gerdts Gunnar , Amann Rudolf
Published: July 2002

We tested new strategies for the isolation of abundant bacteria from coastal North Sea surface waters, which included reducing by several orders of magnitude the concentrations of inorganic N and P compounds in a synthetic seawater medium. Agar plates were resampled over 37 days, and slowly growing colonies were allowed to develop by repeatedly removing all newly formed colonies. A fivefold increase of colonies was observed on plates with reduced nutrient levels, and the phylogenetic composition of the culture collection changed over time, towards members of the
Roseobacter
lineage and other alpha-proteobacteria. Novel gamma-proteobacteria from a previously uncultured but cosmopolitan lineage (NOR5) formed colonies only after 12 days of plate incubation. A time series of German Bight surface waters (January to December 1998) was screened by fluorescence in situ hybridization (FISH) with isolate-specific and general probes. During spring and early summer, a prominent fraction of FISH-detectable bacteria (mean, 51%) were affiliated with the
Cytophaga-Flavobacterium
group (CF) of the
Bacteroidetes
. One
Cytophaga
sp. lineage with cultured representatives formed almost 20% of the CF group. Members of the
Roseobacter
cluster constituted approximately 50% of alpha-proteobacteria, but none of the
Roseobacter
-related isolates formed populations of >1% in the environment. Thus, the readily culturable members of this clade are probably not representative of
Roseobacter
species that are common in the water column. In contrast, members of NOR5 were found at high abundances (>10
5
cells ml
−1
) in the summer plankton. Some abundant pelagic bacteria are apparently able to form colonies on solid media, but appropriate isolation techniques for different species need to be developed.
Substrate Selectivity of a 3-Nitrophenol-Induced Metabolic System in
Pseudomonas putida
2NP8 Transforming Nitroaromatic Compounds into Ammonia under Aerobic Conditions
Authors: Zhao Jian-Shen , Ward Owen P.
Published: July 2002

The 3-nitrophenol-induced enzyme system in cells of
Pseudomonas putida
2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.
Case of Localized Recombination in 23S rRNA Genes from Divergent
Bradyrhizobium
Lineages Associated with Neotropical Legumes
Authors: Parker Matthew A.
Published: July 2002

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of
Bradyrhizobium
sp. nodule bacteria from four papilionoid legumes (
Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis
, and
Desmodium axillare
) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (
Platypodium
and
Machaerium
) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that
Bradyrhizobium
genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to
Bradyrhizobium japonicum
USDA 110, although one ET sampled from
C. javitensis
had a 16S rRNA gene highly similar to that of
Bradyrhizobium elkanii
USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of
B. japonicum
USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of
B. elkanii
USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between
Bradyrhizobium
isolates related to
B. japonicum
versus
B. elkanii
.
2-Bromoethanesulfonate Affects Bacteria in a Trichloroethene-Dechlorinating Culture
Authors: Chiu Pei C. , Lee Minho
Published: July 2002

Long-term exposure to 2-bromoethanesulfonate (BES), an agent known to inhibit methanogenesis, altered the bacterial community structure of an anaerobic enrichment culture that reductively dechlorinated trichloroethene (TCE). BES did not hinder the dechlorination of TCE or other chlorinated ethenes as previously reported, although different intermediates and end products were observed.
Region-Specific Transcriptional Activity in the Genome of
Streptomyces coelicolor
A3(2)
Authors: Gust Bertolt , Spatz Kerstin , Spychaj Alexander , Redenbach Matthias
Published: July 2002

Transcriptional analysis of microbial genomes is an important component of functional genomics. Strategies such as hybridization of labeled total RNA against ordered clone libraries or differential-display approaches have already been carried out to identify expressed genes. We describe here an additional method which applies subtractive hybridization between genome-specific DNA and total RNA followed by a PCR approach to identify expressed microbial genes. With the new strategy, the expression of genes in the terminal regions of the linear
Streptomyces coelicolor
A3(2) chromosome and the accessory linear plasmid SCP1 was analyzed. The results indicate that the method is useful for the identification of expressed genes in actinomycetes and other microbial systems. We demonstrate for the first time that at least 24 genes in the chromosome end regions (silent regions) of
S. coelicolor
are actively expressed. In addition, several expressed SCP1 genes were identified, including a gene which shows high similarity to microbial
dnaN
genes and which seems to play a role in SCP1 maintenance.
Rapid Evolution of Novel Traits in Microorganisms
Authors: Selifonova Olga , Valle Fernando , Schellenberger Volker
Published: July 2002

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.
Use of an Intergenic Region in
Pseudomonas syringae
pv. Syringae B728a for Site-Directed Genomic Marking of Bacterial Strains for Field Experiments
Authors: Hirano Susan S. , Willis David K. , Clayton Murray K. , Upper Christen D.
Published: July 2002

To construct differentially-marked derivatives of our model wild-type strain,
Pseudomonas syringae
pv. syringae B728a (a causal agent of bacterial brown spot disease in snap bean plants), for field experiments, we selected a site in the
gacS-cysM
intergenic region for site-directed insertion of antibiotic resistance marker cassettes. In each of three field experiments, population sizes of the site-directed chromosomally marked B728a derivatives in association with snap bean plants were not significantly different from that of the wild-type strain. Inserts of up to 7 kb of DNA in the intergenic region did not measurably affect fitness of B728a in the field. The site is useful for site-directed genomic insertions of single copies of genes of interest.
Growth Patterns of Two Marine Isolates: Adaptations to Substrate Patchiness?
Authors: Pernthaler Annelie , Pernthaler Jakob , Eilers Heike , Amann Rudolf
Published: July 2002

During bottle incubations of heterotrophic marine picoplankton, some bacterial groups are conspicuously favored. In an earlier investigation bacteria of the genus
Pseudoalteromonas
rapidly multiplied in substrate-amended North Sea water, whereas the densities of
Oceanospirillum
changed little (H. Eilers, J. Pernthaler, and R. Amann, Appl. Environ. Microbiol. 66:4634–4640, 2000). We therefore studied the growth patterns of two isolates affiliating with
Pseudoalteromonas
and
Oceanospirillum
in batch culture. Upon substrate resupply,
Oceanospirillum
lagged threefold longer than
Pseudoalteromonas
but reached more than fivefold-higher final cell density and biomass. A second, mobile morphotype was present in the starved
Oceanospirillum
populations with distinctly greater cell size, DNA and protein content, and 16S rRNA concentration. Contrasting cellular ribosome concentrations during stationary phase suggested basic differences in the growth responses of the two strains to a patchy environment. Therefore, we exposed the strains to different modes of substrate addition. During cocultivation on a single batch of substrates, the final cell densities of
Oceanospirillum
were reduced three times as much as those
Pseudoalteromonas
, compared to growth yields in pure cultures. In contrast, the gradual addition of substrates to stationary-phase cocultures was clearly disadvantageous for the
Pseudoalteromonas
population. Different growth responses to substrate gradients could thus be another facet affecting the competition between marine bacteria and may help to explain community shifts observed during enrichments.
Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in
Lactococcus lactis
Authors: Le Loir Y. , Nouaille S. , Commissaire J. , Brétigny L. , Gruss A. , Langella P.
Published: July 2002

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in
Lactococcus lactis
using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP
Nuc
) by that of
L. lactis
protein Usp45 (SP
Usp
) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP
Usp
than when directed by SP
Nuc
. This SP
Usp
effect on Nuc secretion is not due to a better antifolding activity, since SP
Usp
:Nuc precursor proteins display enzymatic activity in vitro, while SP
Nuc
:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in
L. lactis
(Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP
Usp
and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in
L. lactis
.
Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4
Listeria monocytogenes
and Identify Four Distinct Lineages in Serotype 4b
Authors: Tran Huyen L. , Kathariou Sophia
Published: July 2002

Listeria monocytogenes
of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of
Listeria monocytogenes
had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of
Eco
RI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other
L. monocytogenes
strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of
L. monocytogenes
and may facilitate detection and population genetic analysis of specific lineages.
Continuous Steady-State Method Using Tenax for Delivering Tetrachloroethene to Chloro-Respiring Bacteria
Authors: Brennan Rachel A. , Sanford Robert A.
Published: July 2002

Tenax-TA, a solid-phase sorbent, was used as an alternative to hexadecane for continuous delivery of tetrachloroethene (PCE) to
Desulfuromonas
strain BB1, a chloro-respiring microorganism. In both batch and bioreactor configurations, Tenax not only maintained low, steady-state concentrations of PCE in an active culture for several months but also adsorbed the product of dechlorination,
cis
-1,2-dichloroethene, before it approached toxic levels.
Uric Acid Is a Genuine Metabolite of
Penicillium cyclopium
and Stimulates the Expression of Alkaloid Biosynthesis in This Fungus
Authors: Helbig Florian , Steighardt Jörg , Roos Werner
Published: July 2002

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in
Penicillium cyclopium
uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor
l
-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor
l
-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.
Cloning of
rel
from
Listeria monocytogenes
as an Osmotolerance Involvement Gene
Authors: Okada Yumiko , Makino Sou-ichi , Tobe Toru , Okada Nobuhiko , Yamazaki Shouji
Published: July 2002

Transposon insertional mutants of
Listeria monocytogenes
were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named
rel
, was 2,214 bp in length and had very high homology to
relA
of
Bacillus subtilis
, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included
rel
of
L. monocytogenes
was introduced into the mutant. After exposure to methyl glucoside, the
rel
mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in
L. monocytogenes
and that its mechanism is different from that for the accumulation of compatible solutes.
The
AWA1
Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast
Authors: Shimoi Hitoshi , Sakamoto Kazutoshi , Okuda Masaki , Atthi Ratchanee , Iwashita Kazuhiro , Ito Kiyoshi
Published: July 2002

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as
Saccharomyces cerevisiae
. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named
AWA1
and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an
awa1
disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.
Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute
Spiroplasma citri
Authors: Malembic Sylvie , Saillard Colette , Bové Joseph M. , Garnier Monique
Published: July 2002

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of
Spiroplasma citri
in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on
S. citri
glucose metabolism but were as efficient as polyclonal antibodies in inhibiting
S. citri
growth in liquid medium. Inhibition of motility was also observed.
Oral Treatment with
Lactococcus lactis
Expressing
Staphylococcus hyicus
Lipase Enhances Lipid Digestion in Pigs with Induced Pancreatic Insufficiency
Authors: Drouault Sophie , Juste Catherine , Marteau Philippe , Renault Pierre , Corthier Gérard
Published: July 2002

The
Staphylococcus hyicus lip
gene was cloned in
Lactococcus lactis
. Pancreatic insufficiency was induced by ligation of the pancreatic duct in pigs. In pigs who had undergone pancreatic ligation, the coefficient of fat absorption was higher after consumption of lipase-expressing
L. lactis
(91.9% ± 3.7%) than that after consumption of the inactive control strain (78.4% ± 2.4%).
Antibody Response to Lipopolysaccharide in Patients Colonized or Infected with an Endemic Strain of
Acinetobacter
Genomic Species 13 Sensu Tjernberg and Ursing
Authors: Pantophlet Ralph , Seifert Harald , Brade Lore , Brade Helmut
Published: July 2002

The levels of antilipopolysaccharide (anti-LPS) antibodies in patients colonized with an endemic
Acinetobacter
strain were compared to those in patients with bloodstream infections. Seropositivity and seronegativity correlated with positive and negative blood cultures, respectively, indicating that determination of the level of anti-LPS antibodies is useful for diagnosing
Acinetobacter
infections.
Clinical Application of a Dot Blot Test for Diagnosis of Enteric Fever Due to
Salmonella enterica
Serovar Typhi in Patients with Typhoid Fever from Colombia and Peru
Authors: Cardona-Castro Nora , Gotuzzo Eduardo , Rodriguez Monica , Guerra Humberto
Published: July 2002

Clinical application of a dot blot test to detect immunoglobulin G (IgG) (88% sensitivity and specificity) and IgM (12.1% sensitivity and 97% specificity) against flagellar antigen from
Salmonella enterica
serovar Typhi was performed in Peruvian and Colombian patients with typhoid fever. This test can be used as a good predictor of serovar Typhi infection in regions lacking laboratory facilities and in field studies.
Monoclonal Antibody Differentiation of
Mycoplasma mycoides
subsp.
mycoides
Small-Colony Strains Causing Contagious Bovine Pleuropneumonia from Less Important Large-Colony Strains
Authors: Rurangirwa Fred R. , Shompole Patrick S. , Wambugu Anderson N. , McGuire Travis C.
Published: July 2002

Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine
Mycoplasma mycoides
subsp.
mycoides
small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for
M. mycoides
subsp.
mycoides
small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.
Cloning of the Bovine Immunodeficiency Virus
gag
Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay
Authors: Zheng Ling , Swanson Michelle , Liao Jinghua , Wood Charles , Kapil Sanjay , Snider Ron , Loughin Thomas A. , Minocha Harish C.
Published: July 2002

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The
gag
coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from
Escherichia coli
. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.
Cloning and Expression of Immunoreactive Antigens from
Mycobacterium tuberculosis
Authors: Hong Lim Renee Lay , Kiang Tan Li , Fun Lau Wai , Ching Ming Maxey , Chung  , Dunn Roseanne , Phon Too Heng , Chan Lily
Published: July 2002

Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with molecular weights ranging from 16,000 to 58,000, were observed from immunoblots of
Mycobacterium tuberculosis
total lysates screened with sera from individuals with active tuberculosis. These proteins were identified from microsequence analyses, and genes of proteins with the highest homology were PCR amplified and cloned into the pQE30 vector for expression studies. In addition, a 37.5-kDa protein, designated C17, was identified from a phage expression library of
M. tuberculosis
genomic DNA. Preliminary immunoblot assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and extrapulmonary tuberculosis. The overall ranges of sensitivities, specificities, positive predictive values, and negative predictive values for the recombinant antigens were 20 to 58, 88 to 100, 69 to 100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis.
Diagnosis of Human Immunodeficiency Virus Type 1 Infection with Different Subtypes Using Rapid Tests
Authors: Phillips Susan , Granade Timothy C. , Pau Chou-Pong , Candal Debra , Hu Dale J. , Parekh Bharat S.
Published: July 2002

We evaluated six rapid tests for their sensitivity and specificity in diagnosing human immunodeficiency virus type 1 (HIV-1) infection using 241 specimens (172 HIV-1 positive, 69 HIV-1 negative) representing different HIV-1 subtypes (A [
n
= 40], B [
n
= 47], C [
n
= 28], E [
n
= 42], and F [
n
= 7]). HIVCHEK, Multispot, RTD and SeroStrip were 100% sensitive and specific. Capillus failed to identify two of eight subtype C specimens (overall sensitivity of 98.85%), while the SUDS test (the only test approved by the Food and Drug Administration) gave false-positive results for 5 of 69 seronegative specimens (specificity of 93.24%). Our results suggest that although rapid tests perform well in general, it may be prudent to evaluate a rapid test for sensitivity and specificity in a local population prior to its widespread use.
Diagnosis of Granulocytic Ehrlichiosis in Humans by Immunofluorescence Assay
Authors: Brouqui P. , Salvo E. , Dumler J. S. , Raoult D.
Published: July 2002

Serodiagnostic tests are widely available for tick-borne diseases. We evaluated a cell-free antigen of the human granulocytic ehrlichiosis agent. Immunofluorescence assay (IFA) with this antigen is as efficient as with the MRL kit and allows a one-step IFA with other cell-free antigens that is useful when testing sera from patients bitten by ticks.
PCR-Based Detection, Restriction Endonuclease Analysis, and Transcription of
tonB
in
Haemophilus influenzae
and
Haemophilus parainfluenzae
Isolates Obtained from Children Undergoing Tonsillectomy and Adenoidectomy
Authors: Matar Ghassan M. , Chahwan Richard , Fuleihan Nabil , Uwaydah Marwan , Hadi Usamah
Published: July 2002

We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the
tonB
gene of
Haemophilus influenzae
and
Haemophilus parainfluenzae
from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55
H. influenzae
isolates were recovered from different sites of 20 patients, and 32
H. parainfluenzae
isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the
tonB
gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using
Xho
I and
Bgl
II were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the
tonB
gene was detected in both species. It is known to encode a virulent protein, TonB, in
H. influenzae
; however, demonstration of its presence in
H. parainfluenzae
is novel. Nested-PCR and restriction endonuclease analysis have shown that the
tonB
gene is apparently structurally the same in both species, with possible differences that may exist in certain
H. parainfluenzae
isolates. RT-PCR done on selected numbers of
H. influenzae
and
H. parainfluenzae
have shown that the
tonB
gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in
H. parainfluenzae
since it is not needed for heme or heme complexes uptake as with
H. influenzae.
Are the Opsonophagocytic Activities of Antibodies in Infant Sera Measured by Different Pneumococcal Phagocytosis Assays Comparable?
Authors: Väkeväinen Merja , Jansen Wouter , Saeland Eirikur , Jonsdottir Ingileif , Snippe Harm , Verheul Andre , Käyhty Helena
Published: July 2002

Host protection against
Streptococcus pneumoniae
is mainly mediated by opsonin-dependent phagocytosis. Several techniques for measuring opsonophagocytic activity (OPA) of antibodies to
S. pneumoniae
have been standardized and used. These include the viable cell-assay, flow-cytometric assays, and an assay utilizing radiolabeled bacteria. Using these different methods, we measured the OPA of antibodies to
S. pneumoniae
types 6B and 19F from the sera of infants immunized with a pneumococcal conjugate vaccine, PncCRM. Generally, the results obtained by the various techniques correlated well, although serotype-specific differences were found (6B,
r
= 0.78 to 0.95,
P
< 0.001; 19F,
r
= 0.50 to 0.84,
P
< 0.001). The same serotype-specific differences were observed for the relationship between the concentrations of specific immunoglobulin G antibodies measured by enzyme immunoassay and the OPA. Since the sensitivities of the OPA assays differed, the most prominent discrepancies between the techniques were found at low antibody concentrations.