bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex PCR for Detection and Identification of Lactococcal Bacteriophages
Authors: Labrie Steve , Moineau Sylvain
Published: July 2002

Three genetically distinct groups of
Lactococcus lactis
phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (
mcp
) as the target. The
mcp
sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five
mcp
genes. The gene of the only major structural protein (
msp
) was selected as a target for the detection of 936-related phages. The
msp
sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six
msp
genes revealed an 82.2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to
orfF17-orf18
of phage r1t and
orf20-orf21
of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10
4
to 10
7
PFU/ml and was 10
3
to 10
5
PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.
Nitrile Hydratase and Amidase from
Rhodococcus rhodochrous
Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles
Authors: Tauber M. M. , Cavaco-Paulo A. , Robra K.-H. , Gübitz G. M.
Published: July 2002

Rhodococcus rhodochrous
NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein
−1
) and amidase activity (38.4 nkat mg of protein
−1
) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the
R. rhodochrous
enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (
K/S
value) for C.I. Basic Blue 9.
Molecular and Ecological Evidence for Species Specificity and Coevolution in a Group of Marine Algal-Bacterial Symbioses
Authors: Ashen Jon B. , Goff Lynda J.
Published: July 2002

The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus
Prionitis
(Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of
Prionitis
,
P. lanceolata
(from central California) and
P. decipiens
(from Peru). This investigation reports gall formation on a third related host,
Prionitis filiformis
. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the
Roseobacter
grouping of the α subclass of the division
Proteobacteria
(99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in
Prionitis
gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.
Characterization of the Minimal Replicon of a Cryptic
Deinococcus radiodurans
SARK Plasmid and Development of Versatile
Escherichia coli-D. radiodurans
Shuttle Vectors
Authors: Meima Rob , Lidstrom Mary E.
Published: July 2002

The nucleotide sequence of a 12-kb fragment of the cryptic
Deinococcus radiodurans
SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for
Deinococcus
. Annotation of the sequence revealed 12 possible open reading frames. Among these are the
repU
and
resU
genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in
Escherichia coli
. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of
repU
expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from
D. radiodurans
strain SARK is similar to that for plasmids found in
Thermus
but not to that for the 45.7-kb plasmid found in
D. radiodurans
strain R1. The minimal region required for autonomous replication in
D. radiodurans
was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for
E. coli
and
D. radiodurans
. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing
lacZ
and a
Deinococcus
promoter was shown to efficiently express LacZ.
Response of a Soil Bacterial Community to Grassland Succession as Monitored by 16S rRNA Levels of the Predominant Ribotypes
Authors: Felske Andreas , Wolterink Arthur , Van Lis Robert , De Vos Willem M. , Akkermans Antoon D. L.
Published: July 2002

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented
Bacillus
and members of the
Acidobacterium
cluster and the α subclass of the class
Proteobacteria
. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the
Bacteria
probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant
Lolium perenne
population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.
Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization
Authors: Rudi Knut , Skulberg Olav M. , Skulberg Randi , Jakobsen Kjetill S.
Published: July 2002

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (
Nostoc
,
Microcystis
, and
Planktothrix
species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.
Role for Outer Membrane Cytochromes OmcA and OmcB of
Shewanella putrefaciens
MR-1 in Reduction of Manganese Dioxide
Authors: Myers Judith M. , Myers Charles R.
Published: July 2002

Shewanella putrefaciens
MR-1 can use a wide variety of terminal electron acceptors for anaerobic respiration, including certain insoluble manganese and iron oxides. To examine whether the outer membrane (OM) cytochromes of MR-1 play a role in Mn(IV) and Fe(III) reduction, mutants lacking the OM cytochrome OmcA or OmcB were isolated by gene replacement. Southern blotting and PCR confirmed replacement of the
omcA
and
omcB
genes, respectively, and reverse transcription-PCR analysis demonstrated loss of the respective mRNAs, whereas mRNAs for upstream and downstream genes were retained. The
omcA
mutant (OMCA1) resembled MR-1 in its growth on trimethylamine
N
-oxide (TMAO), dimethyl sulfoxide, nitrate, fumarate, thiosulfate, and tetrathionate and its reduction of nitrate, nitrite, ferric citrate, FeOOH, and anthraquinone-2,6-disulfonic acid. Similarly, the
omcB
mutant (OMCB1) grew on fumarate, nitrate, TMAO, and thiosulfate and reduced ferric citrate and FeOOH. However, OMCA1 and OMCB1 were 45 and 75% slower than MR-1, respectively, at reducing MnO
2
. OMCA1 lacked only OmcA. While OMCB1 lacked OmcB, other OM cytochromes were also missing or markedly depressed. The total cytochrome content of the OM of OMCB1 was less than 15% of that of MR-1. Western blots demonstrated that OMCB1 still synthesized OmcA, but most of it was localized in the cytoplasmic membrane and soluble fractions rather than in the OM. OMCB1 had therefore lost the ability to properly localize multiple OM cytochromes to the OM. Together, the results suggest that the OM cytochromes of MR-1 participate in the reduction of Mn(IV) but are not required for the reduction of Fe(III) or other electron acceptors.
Effect of Water Activities of Heating and Recovery Media on Apparent Heat Resistance of
Bacillus cereus
Spores
Authors: Coroller Louis , Leguérinel Ivan , Mafart Pierre
Published: July 2002

Spores of
Bacillus cereus
were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.
The Green Fluorescent Protein Gene Functions as a Reporter of Gene Expression in
Phanerochaete chrysosporium
Authors: Ma Biao , Mayfield Mary B. , Gold Michael H.
Published: July 2002

The enhanced green fluorescent protein (GFP) gene (
egfp
) was used as a reporter of gene expression driven by the glyceraldehyde-
p
-dehydrogenase (
gpd
) gene promoter and the manganese peroxidase isozyme 1 (
mnp1
) gene promoter in
Phanerochaete chrysosporium
. Four different constructs were prepared. pUGGM3′ and pUGiGM3′ contain the
P. chrysosporium gpd
promoter fused upstream of the
egfp
coding region, and pUMGM3′ and pUMiGM3′ contain the
P. chrysosporium mnp1
promoter fused upstream of the
egfp
gene. In all constructs, the
egfp
gene was followed by the
mnp1
gene 3′ untranslated region. In pUGGM3′ and pUMGM3′, the promoters were fused directly with
egfp
, whereas in pUGiGM3′ and pUMiGM3′, following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the
gpd
gene were inserted at the 5′ end of the
egfp
gene. All constructs were ligated into a plasmid containing the
ura1
gene of
Schizophyllum commune
as a selectable marker and were used to transform a Ural1 auxotrophic strain of
P. chrysosporium
to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5′ of the
egfp
gene (pUGiGM3′ and pUMiGM3′) exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5′ intron resulted in more
egfp
RNA than was found in transformants carrying an intronless
egfp
. These results suggest that the presence of a 5′ intron affects the expression of the
egfp
gene in
P. chrysosporium
. The expression of GFP in the transformants carrying pUMiGM3′ paralled the expression of endogenous
mnp
with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying
cis
-acting elements in the
mnp1
gene promoter.
Specificity and Mode of Action of the Antifungal Fatty Acid
cis
-9-Heptadecenoic Acid Produced by
Pseudozyma flocculosa
Authors: Avis Tyler J. , Bélanger Richard R.
Published: July 2002

cis
-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent
Pseudozyma flocculosa
, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by
P. flocculosa
, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.
Biotransformation of
d
-Limonene to (+)
trans
-Carveol by Toluene-Grown
Rhodococcus opacus
PWD4 Cells
Authors: Duetz Wouter A. , Fjällman Ann H. M. , Ren Shuyu , Jourdat Catherine , Witholt Bernard
Published: July 2002

The toluene-degrading strain
Rhodococcus opacus
PWD4 was found to hydroxylate
d
-limonene exclusively in the 6-position, yielding enantiomerically pure (+)
trans
-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+)
trans
-carveol formation was 14.7 U (g of cells [dry weight])
−1
, and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the
d
-limonene conversion. Glucose-grown cells did not form any
trans
-carveol from
d
-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (
Rhodococcus globerulus
PWD8) had a lower specific activity but was found to oxidize most of the formed
trans
-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.
Duplication of Hemolysin Genes in a Virulent Isolate of
Vibrio harveyi
Authors: Zhang X.-H. , Meaden P. G. , Austin B.
Published: July 2002

Vibrio harveyi
VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated
vhhA
and
vhhB
), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene. Both genes from VIB 645 were cloned and sequenced. The open reading frames (ORFs) of
vhhA
and
vhhB
shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues. The VHH protein shows homology to the lecithinase of
V. mimicus
and
V. cholerae
. Transformants of
Escherichia coli
containing the ORF of either
vhhA
or
vhhB
displayed weak hemolytic activity in rainbow trout blood agar. The hemolytic activity was very high when the ORF of
vhhB
was cloned in
E. coli
together with the native promoter. Surprisingly, the level of
vhh
-specific RNA transcript produced by VIB 645 was found to be very low. We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of the
vhh
gene.
Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with
Rhodococcus
sp. Strain DN22
Authors: Fournier Diane , Halasz Annamaria , Spain Jim , Fiurasek Petr , Hawari Jalal
Published: July 2002

Rhodococcus
sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO
2
−
) (30%), nitrous oxide (N
2
O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [
15
N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N
2
O with two molecular mass ions: one at 44 Da, corresponding to
14
N
14
NO, and the second at 45 Da, corresponding to
15
N
14
NO. The nonlabeled N
2
O could be formed only from -NO
2
, whereas the
15
N-labeled one was presumed to originate from a nitramine group (
15
N-
14
NO
2
) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C
2
H
5
N
3
O
3
. When the experiment was repeated with ring-labeled [
15
N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-
14
C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in
14
CO
2
(mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO
2
and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.
Identification of the 2-Methylcitrate Pathway Involved in the Catabolism of Propionate in the Polyhydroxyalkanoate-Producing Strain
Burkholderia sacchari
IPT101
T
and Analysis of a Mutant Accumulating a Copolyester with Higher 3-Hydroxyvalerate Content
Authors: Brämer C. O. , Silva L. F. , Gomez J. G. C. , Priefert H. , Steinbüchel A.
Published: July 2002

Burkholderia sacchari
IPT101
T
induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The
prp
locus of
B. sacchari
IPT101
T
is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes (
prpR
,
prpB
,
prpC
,
acnM
, and ORF5) exhibited identity to genes located in the
prp
loci of other gram-negative bacteria.
prpC
encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da.
prpB
encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101
T
and were heterologously expressed in
Escherichia coli.
The
acnM
gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in
Ralstonia eutropha
HF39, are also located in the
prp
locus. The translational product of ORF1 (
prpR
) had a calculated molecular mass of 70,598 Da and is a putative regulator of the
prp
cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the
prp
locus of
B. sacchari
, and these open reading frames have not been found in any other
prp
operon yet. In summary, the organization of the
prp
genes of
B. sacchari
is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a
B. sacchari
mutant since it was revealed in this study that the mutant is defective in
prpC
.
Nitrite as a Stimulus for Ammonia-Starved
Nitrosomonas europaea
Authors: Laanbroek Hendrikus J. , Bär-Gilissen Marie-José , Hoogveld Hans L.
Published: July 2002

Ammonia-starved cells of
Nitrosomonas europaea
are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of
N. europaea
starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.
Photosynthetic Apparatus in
Roseateles depolymerans
61A Is Transcriptionally Induced by Carbon Limitation
Authors: Suyama Tetsushi , Shigematsu Toru , Suzuki Toshihiko , Tokiwa Yutaka , Kanagawa Takahiro , Nagashima Kenji V. P. , Hanada Satoshi
Published: July 2002

Production of a photosynthetic apparatus in
Roseateles depolymerans
61A, a recently discovered freshwater β-
Proteobacterium
showing characteristics of aerobic phototrophic bacteria, was observed when the cells were subjected to a sudden decrease in carbon sources (e.g., when cells grown with 0.1 to 0.4% Casamino Acids were diluted or transferred into medium containing ≤0.04% Casamino Acids). Accumulation of bacteriochlorophyll (BChl)
a
was observed in the presence of oxygen and was enhanced under semiaerobic conditions (2% oxygen) but was reduced in the presence of light. Similarly to what has been reported regarding some aerobic phototrophic bacteria belonging to the α subclass of the
Proteobacteria
, viability of the cells in the carbon source-free medium was prolonged under aerobic-light (10 W m
−2
) conditions, possibly due to photosynthetic energy conversion, but was not prolonged under aerobic-dark conditions. The
puf
operon, which encodes most of the apoproteins of light-harvesting and reaction center complexes, was sequenced, and the effect of changes in Casamino Acids concentrations, oxygen, and light on its expression was estimated by the accumulation of its mRNA. The expression of the
puf
operon was induced by the decrease in carbon sources, similarly to what was observed for the accumulation of BChl
a
under aerobic and semiaerobic conditions (≥0.2% O
2
), and was reduced in the presence of light. Transcription of the
R. depolymerans puf
operon is considered to be controlled by changes in carbon nutrients in addition to oxygen tension and light intensity.
Use of Recombinant Cellulose-Binding Domains of
Trichoderma reesei
Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa
Authors: Linder Markus , Winiecka-Krusnell Jadwiga , Linder Ewert
Published: July 2002

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in
Acanthamoeba
, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from
Trichoderma reesei
cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of
Acanthamoeba
strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of
Giardia intestinalis
,
Entamoeba dispar
, or
Pneumocystis carinii
. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as
Acanthamoeba
spp. Five isolates of
Hartmannella
and seven isolates of
Naegleria
tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of
Acanthamoeba
spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.
Negative Effect of High pH on Biocidal Efficacy of Copper and Silver Ions in Controlling
Legionella pneumophila
Authors: Lin Yu-sen E. , Vidic Radisav D. , Stout Janet E. , Yu Victor L.
Published: July 2002

Copper-silver (Cu-Ag) ionization has effectively controlled
Legionella
spp. in the hot water systems of numerous hospitals. However, it was ineffective at controlling
Legionella
in one Ohio hospital despite the confirmation of adequate total concentrations of copper and silver ions. The pH of the water at this hospital was found to be 8.5 to 9.0. The purpose of this study was to investigate the impact of pH and other water quality parameters, including alkalinity (HCO
3
−
), hardness (Ca
2+
and Mg
2+
), and amount of dissolved organic carbon (DOC), on the control of
Legionella
by Cu-Ag ionization. Initial concentrations of
Legionella
and copper and silver ions used in batch experiments were 3 × 10
6
CFU/ml and 0.4 and 0.08 mg/liter, respectively. Changes in bicarbonate ion concentration (50, 100, and 150 mg/liter), water hardness (Ca
2+
at 50 and 100 mg/liter; Mg
2+
at 40 and 80 mg/liter), and level of DOC (0.5 and 2 mg/liter) had no significant impact on the efficacy of copper and silver ions in killing
Legionella
at a neutral pH. When the pH was elevated to 9 in these experiments, copper ions achieved only a 10-fold reduction in the number of
Legionella
organisms in 24 h, compared to a millionfold decrease at pH 7.0. Silver ions were able to achieve a millionfold reduction in 24 h at all ranges of water quality parameters tested. Precipitation of insoluble copper complexes was observed at a pH above 6.0. These results suggest that pH may be an important factor in the efficacy of copper-silver ionization in controlling
Legionella
in water systems.
Functional Analysis of
Sinorhizobium meliloti
Genes Involved in Biotin Synthesis and Transport
Authors: Entcheva Plamena , Phillips Donald A. , Streit Wolfgang R.
Published: July 2002

External biotin greatly stimulates bacterial growth and alfalfa root colonization by
Sinorhizobium meliloti
strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the
S. meliloti
genome database in combination with complementation tests of
Escherichia coli
biotin auxotrophs indicate that biotin synthesis probably is limited in
S. meliloti
1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only
bioB
,
bioF
, and
bioH
were demonstrably functional in complementation tests with known
E. coli
mutants. No sequence or complementation evidence was found for
bioA
,
bioC
,
bioD
, or
bioZ
. In contrast to other microorganisms, the
S. meliloti bioB
and
bioF
genes are not localized in a biotin synthesis operon, but
bioB
is cotranscribed with two genes coding for ABC transporter-like proteins, designated here
bioM
and
bioN.
Mutations in
bioM
and
bioN
eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that
S. meliloti
normally grows on exogenous biotin using
bioM
and
bioN
to conserve biotin assimilated from external sources.
Variations in the Membrane Fatty Acid Composition of Resistant or Susceptible
Leuconostoc
or
Weissella
Strains in the Presence or Absence of Mesenterocin 52A and Mesenterocin 52B Produced by
Leuconostoc mesenteroides
subsp.
mesenteroides
FR52
Authors: Limonet Maxime , Revol-Junelles Anne-Marie , Millière Jean-Bernard
Published: July 2002

Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by
Leuconostoc mesenteroides
subsp.
mesenteroides
FR 52. Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity. Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity. Four
Leuconostoc
and
Weissella
wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins. Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media. These resistant strains were at least 30 times more resistant than the wild-type strains. No cross-resistance to Mes52A and Mes52B was observed in these strains. No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth. Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition. When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane. Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents.
Weissella paramesenteroides
DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.
Genetic and Biochemical Characterization of Glycerol Uptake in
Mycoplasma mycoides
subsp.
mycoides
SC: Its Impact on H
2
O
2
Production and Virulence
Authors: Vilei Edy M. , Frey Joachim
Published: July 2002

Highly virulent strains of
Mycoplasma mycoides
subsp.
mycoides
SC belonging to the African cluster contain an operon with the genes
gtsA
,
gtsB
, and
gtsC
, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-
14
C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks
gtsB
and
gtsC
, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake.
l
-α-Glycerophosphate was not transported by
M. mycoides
subsp.
mycoides
SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H
2
O
2
. Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H
2
O
2
was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains.
M. mycoides
subsp.
mycoides
SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.
T-Cell-Mediated Immune Responses in Patients with Cutaneous or Mucosal Leishmaniasis: Long-Term Evaluation after Therapy
Authors: Da-Cruz Alda Maria , Bittar Rita , Mattos Marise , Oliveira-Neto Manuel P. , Nogueira Ricardo , Pinho-Ribeiro Vanessa , Azeredo-Coutinho Rilza Beatriz , Coutinho Sergio G.
Published: July 2002

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4+and CD8+Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4+than CD8+T cells. In CL, the healing process was associated with a decrease of CD4+and an increase of CD8+, leading to similar CD4+and CD8+proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4+/CD8+ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-γ) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-γ and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-γ, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4+Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations.
Defective Induction of Interleukin-12 in Human Monocytes by Germ-Tube Forms ofCandida albicans
Authors: Chiani Paola , Bromuro Carla , Torosantucci Antonella
Published: July 2002

Yeast (Y) to germ-tube (GT) transition ofCandida albicansis considered a putative virulence trait. On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms ofC. albicans. Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-γ), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 ± 65 and 185 ± 80 pg/ml, respectively [mean ± standard deviation in four independent experiments]). In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-γ (30 ± 10 pg/ml in the four independent experiments above). Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells ofC. albicanswere not phagocytized by the human monocytes. Importantly, however, Y and GT cells were equally killed by the monocytes. Thus, the virulence trait attributed to the Y-GT transition ofC. albicansmight also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.
Lectin Site Interaction with Capsular Polysaccharide Mediates Nonimmune Phagocytosis of Type III Group B Streptococci
Authors: Albanyan Esam A. , Edwards Morven S.
Published: July 2002

Group BStreptococcus(GBS) causes substantial morbidity but most individuals exposed to the organism remain healthy. These experiments tested the hypothesis that engagement of the complement receptor 3 (CR3) lectin site would effectively trigger neutrophil-mediated phagocytosis of complement-opsonized type III GBS by nonimmune human sera. Using an opsonophagocytosis assay, saccharides identified as interacting with the CR3 lectin site effectively inhibited neutrophil-mediated killing of type III, strain COH1. Fructose, which does not interact with the lectin site, promoted significantly less inhibition of opsonophagocytosis. Saccharide-mediated inhibition was reversed in a dose-related fashion by addition of type III, GBS capsular polysaccharide-specific immunoglobulin G. When capsule-deficient or asialo mutant type III strains were employed, the lectin site was not required. Structurally defined GBS serotypes with a side chain at least two sugars in length engaged the lectin site, andN-acetyld-glucosamine was not a required component monosaccharide. Intact type III capsular polysaccharide interacted significantly more efficiently with the lectin site than did oligosaccharides representing approximately 5 or 20 repeating units, respectively. Taken together, these experiments indicate that interaction of type III GBS capsular polysaccharide with the lectin site of CR3 effects phagocytosis of these organisms by nonimmune serum. Use of this mechanism of innate immunity provides a potential explanation for the infrequency with which susceptible individuals exposed to type III GBS develop invasive infection.
Genetic Vaccination against Malaria Infection by Intradermal and Epidermal Injections of a Plasmid Containing the Gene Encoding thePlasmodium bergheiCircumsporozoite Protein
Authors: Weiss Richard , Leitner Wolfgang W. , Scheiblhofer Sandra , Chen Defeng , Bernhaupt Andrea , Mostböck Sven , Thalhamer Josef , Lyon Jeffrey A.
Published: July 2002

The circumsporozoite protein (CSP) from the surface of sporozoite stagePlasmodiumsp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encodingPlasmodium bergheiCSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced byP. bergheiCSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.
Protective and Nonprotective Epitopes from Amino Termini of M Proteins from Australian Aboriginal Isolates and Reference Strains of Group A Streptococci
Authors: Brandt Evelyn R. , Teh Terrence , Relf Wendy A. , Hobb Rhonda I. , Good Michael F.
Published: July 2002

The M protein is the primary vaccine candidate to prevent group A streptococcal (GAS) infection and the subsequent development of rheumatic fever (RF). However, the large number of serotypes have made it difficult to design a vaccine against all strains. We have taken an approach of identifying amino-terminal M protein epitopes from GAS isolates that are highly prevalent in GAS-endemic populations within the Northern Territory (NT) of Australia. Australian Aboriginals in the NT experience the highest incidence of RF worldwide. To develop a vaccine for this population, 39 peptides were synthesized, representing the amino-terminal region of the M protein from endemic GAS. Mice immunized with these peptides covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant raised high-titer antibodies. Over half of these sera reduced bacterial colony counts by >80% against the homologous isolate of GAS. Seven of the peptide antisera also cross-reacted with at least three other heterologous peptides by enzyme-linked immunosorbent assay. Antiserum to one peptide, BSA101–28, could recognize six other peptides, and five of these peptides could inhibit opsonization mediated by BSA101–28antiserum. Cross-opsonization studies showed that six of these sera could opsonize at least one heterologous isolate of GAS. These data reveal vaccine candidates specific to a GAS-endemic area and show the potential of some to cross-opsonize multiple isolates of GAS. This information will be critical when considering which epitopes may be useful in a multiepitope vaccine to prevent GAS infection.
Dual Role of Signaling Pathways Leading to Ca2+and Cyclic AMP Elevation in Host Cell Invasion byTrypanosoma cruzi
Authors: Caler Elisabet V. , Morty Rory E. , Burleigh Barbara A. , Andrews Norma W.
Published: July 2002

Cell invasion by the protozoan parasiteTrypanosoma cruziinvolves activation of host signaling pathways and the recruitment and fusion of lysosomes at the parasite entry site. A major signaling pathway regulating invasion of fibroblasts, epithelial cells, and myoblasts involves mobilization of Ca2+from intracellular stores and requires the activity of aT. cruziserine peptidase, oligopeptidase B (OPB). Deletion of the OPB gene results in a marked defect in trypomastigote virulence, consistent with a greatly reduced cell invasion capacity. Here we show that uptake by macrophages, on the other hand, is largely independent of OPB expression and sensitive to inhibition of by cytochalasin D. The residual invasion capacity of OPBnull trypomastigotes in fibroblasts still involves lysosome recruitment, although in a significantly delayed fashion. Transient elevations in intracellular Ca2+concentrations were observed in host cells exposed to both wild-type and OPBnull trypomastigotes, but the signals triggered by the mutant parasites were less vigorous and delayed. The capacity of triggering elevation in host cell cyclic AMP (cAMP), however, was unaltered in OPBnull trypomastigotes. Modulation in cAMP levels preferentially affected the residual cell invasion capacity of OPBnull parasites, suggesting that this signaling pathway can play a dominant role in promoting cell invasion in the absence of the major OPB-dependent pathway.