bluuebunny/crossref_metadata_2025 huggingface dataset:
Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by
Rhodococcus
sp. Strain DN22
Authors: Bhushan Bharat , Trott Sandra , Spain Jim C. , Halasz Annamaria , Paquet Louise , Hawari Jalal
Published: March 2003

A unique metabolite with a molecular mass of 119 Da (C
2
H
5
N
3
O
3
) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by
Rhodococcus
sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with
Rhodococcus
sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of
Rhodococcus
sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and
Rhodococcus
sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by
Rhodococcus
sp. strain DN22.
Purification and Characterization of Brochocin A and Brochocin B(10-43), a Functional Fragment Generated by Heterologous Expression in
Carnobacterium piscicola
Authors: Garneau Sylvie , Ference Claire A. , van Belkum Marco J. , Stiles Michael E. , Vederas John C.
Published: March 2003

Brochothrix campestris
ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of
Clostridium botulinum
. An improved purification method was developed for BrcC, in which
n
-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by
B. campestris
as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in
Carnobacterium piscicola
LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by
C. piscicola
as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high β-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of
brcA
and
brcB
were also constructed in
Escherichia coli
, but these clones only produced multiple fragments of the desired peptides with little or no activity.
Community Structure of Ammonia-Oxidizing Bacteria within Anoxic Marine Sediments
Authors: Freitag Thomas E. , Prosser James I.
Published: March 2003

The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by
Nitrosomonas
-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of
Planctomycetales-Verrucomicrobia
and β- and γ-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR- and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for
Planctomycetales-Verrucomicrobia
and β-proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for γ-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of β-proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of
Nitrosospira
cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized β-proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.
Expression of Chlorocatechol 1,2-Dioxygenase and Chlorocatechol 2,3-Dioxygenase Genes in Chlorobenzene-Contaminated Subsurface Samples
Authors: Alfreider Albin , Vogt Carsten , Babel Wolfgang
Published: March 2003

In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H
2
O
2
. Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from
Pseudomonas chlororaphis
RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene
cbzE
from
Pseudomonas putida
GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.
New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in
Lactobacillus gasseri
Authors: Neu T. , Henrich B.
Published: March 2003

Derivatives of a cryptic plasmid from
Lactobacillus curvatus
showed temperature-sensitive replication in thermophilic lactobacilli. The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences. pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35°C, whereas replication is efficiently shut down at 42°C. To demonstrate the functionality of pTN1, the signal transduction genes (
nisRK
) of the nisin-controlled expression system were integrated downstream of the
pepN
gene into the chromosome of
Lactobacillus gasseri
. In the resulting strain, UKLbg1, expression of
nisRK
was likely driven by cotranscription with
pepN
and enabled nisin-dependent induction of a fusion of a reporter gene (
pepI
) to the
nisA
promoter. The induction rates were correlated with the amount of nisin used, and maximum
pepI
expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.
A Real-Time PCR Assay for the Detection of
Campylobacter jejuni
in Foods after Enrichment Culture
Authors: Sails Andrew D. , Fox Andrew J. , Bolton Frederick J. , Wareing David R. A. , Greenway David L. A.
Published: March 2003

A real-time PCR assay was developed for the quantitative detection of
Campylobacter jejuni
in foods after enrichment culture. The specificity of the assay for
C. jejuni
was demonstrated with a diverse range of
Campylobacter
species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect
C. jejuni
in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free
Campylobacter
enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to
Campylobacter
charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive
Campylobacter
isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for
C. jejuni
by either method, with 57 samples positive for
C. jejuni
by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of
C. jejuni
in foods and is an important model for other food-borne pathogens.
Detection and Typing of
Campylobacter jejuni
and
Campylobacter coli
and Analysis of Indicator Organisms in Three Waterborne Outbreaks in Finland
Authors: Hänninen Marja-Liisa , Haajanen H. , Pummi T. , Wermundsen K. , Katila M.-L. , Sarkkinen H. , Miettinen I. , Rautelin H.
Published: March 2003

Waterborne outbreaks associated with contamination of drinking water by
Campylobacter jejuni
are rather common in the Nordic countries Sweden, Norway, and Finland, where in sparsely populated districts groundwater is commonly used without disinfection. Campylobacters,
Escherichia coli
, or other coliforms have rarely been detected in potential sources. We studied three waterborne outbreaks in Finland caused by
C. jejuni
and used sample volumes of 4,000 to 20,000 ml for analysis of campylobacters and sample volumes of 1 to 5,000 ml for analysis of coliforms and
E. coli
, depending on the sampling site. Multiple samples obtained from possible sources (water distribution systems and environmental water sources) and the use of large sample volumes (several liters) increased the chance of detecting the pathogen
C. jejuni
in water. Filtration of a large volume (1,000 to 2,000 ml) also increased the rate of detection of coliforms and
E. coli
. To confirm the association between drinking water contamination and illness, a combination of Penner serotyping and pulsed-field gel electrophoresis (digestion with
Sma
I and
Kpn
I) was found to be useful. This combination reliably verified similarity or dissimilarity of
C. jejuni
isolates from patient samples, from drinking water, and from other environmental sources, thus confirming the likely reservoir of an outbreak.
Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on
Escherichia coli
Authors: Knietsch Anja , Waschkowitz Tanja , Bowien Susanne , Henne Anke , Daniel Rolf
Published: March 2003

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C
2
to C
4
) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000
Escherichia coli
strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive
E. coli
clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of
E. coli
were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (
orf12
,
orf14
, and
orf22
) encoded novel alcohol dehydrogenases of different types, four (
orf5
,
sucB
,
fdhD
, and
yabF
) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (
glpK
) encoded a putative glycerol kinase, and one (
orf1
) encoded a protein which showed no similarity to any other known gene product.
Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids
Authors: Peng Xue , Misawa Norihiko , Harayama Shigeaki
Published: March 2003

Thirty-four thermophilic
Bacillus
sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(
p
-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.
Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in
Drosophila melanogaster
Authors: Anbutsu Hisashi , Fukatsu Takema
Published: March 2003

The endosymbiotic bacteria
Spiroplasma
spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in
Drosophila
species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host
Drosophila melanogaster
by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 × 10
8
dnaA
copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 × 10
9
dnaA
copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 × 10
−5
for NSRO and 3 × 10
−4
for NSRO-A.
Evaluation of Genetic Diversity among
Pseudomonas citronellolis
Strains Isolated from Oily Sludge-Contaminated Sites
Authors: Bhattacharya Dhruva , Sarma Priyangshu M. , Krishnan S. , Mishra Sanjeet , Lal Banwari
Published: March 2003

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing.
Pseudomonas citronellolis
predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of
P. citronellolis
were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of
P. citronellolis
were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of
P. citronellolis
.
Isolation of Novel Ultramicrobacteria Classified as Actinobacteria from Five Freshwater Habitats in Europe and Asia
Authors: Hahn Martin W. , Lünsdorf Heinrich , Wu Qinglong , Schauer Michael , Höfle Manfred G. , Boenigk Jens , Stadler Peter
Published: March 2003

We describe the first freshwater members of the class
Actinobacteria
that have been isolated. Nine ultramicro-size (<0.1 μm
3
) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 μm
3
and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class
Actinobacteria
and the family
Microbacteriaceae
. The previously described species with the highest levels of sequence similarity are
Clavibacter michiganensis
and
Rathayibacter tritici
, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate
Ochromonas
sp. strain DS.
Survival of Bacterial Indicator Species and Bacteriophages after Thermal Treatment of Sludge and Sewage
Authors: Mocé-Llivina Laura , Muniesa Maite , Pimenta-Vale Hugo , Lucena Francisco , Jofre Juan
Published: March 2003

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80°C, and the sewage was heated at 60°C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting
Bacteroides fragilis
were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than
Salmonella choleraesuis
, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of
Escherichia coli
and
Salmonella
.
Use of a Packed-Column Bioreactor for Isolation of Diverse Protease-Producing Bacteria from Antarctic Soil
Authors: Wery Nathalie , Gerike Ursula , Sharman Ajay , Chaudhuri Julian B. , Hough David W. , Danson Michael J.
Published: March 2003

Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10°C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45°C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: α-, β-, and γ-
Proteobacteria
; the
Flexibacter
-
Cytophaga
-
Bacteroides
group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new species of the genera
Chryseobacterium
and
Massilia
. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.
ASynechococcusPglnA::luxABFusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria
Authors: Gillor Osnat , Harush Ayelet , Hadas Ora , Post Anton F. , Belkin Shimshon
Published: March 2003

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, PglnA, fromSynechococcussp. strain PCC7942 to theluxABluciferase-encoding genes of the bioluminescent bacteriumVibrio harveyi. The resulting construct was introduced into a neutral site on theSynechococcuschromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.
Effects of Light on the Microcystin Content of
Microcystis
Strain PCC 7806
Authors: Wiedner Claudia , Visser Petra M. , Fastner Jutta , Metcalf James S. , Codd Geoffrey A. , Mur Luuc R.
Published: March 2003

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by
Microcystis
strain PCC 7806 were studied in continuous cultures.
Microcystis
strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m
−2
s
−1
on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell
−1
and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m
−2
s
−1
, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m
−2
s
−1
than at 10 μmol of photons m
−2
s
−1
and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of
Microcystis
strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.
Relationship between Spatial and Genetic Distance in
Agrobacterium
spp. in 1 Cubic Centimeter of Soil
Authors: Vogel J. , Normand P. , Thioulouse J. , Nesme X. , Grundmann G. L.
Published: March 2003

The spatial and genetic unit of bacterial population structure is the clone. Surprisingly, very little is known about the spread of a clone (spatial distance between clonally related bacteria) and the relationship between spatial distance and genetic distance, especially at very short scale (microhabitat scale), where cell division takes place.
Agrobacterium
spp. Biovar 1 was chosen because it is a soil bacterial taxon easy to isolate. A total of 865 microsamples 500 μm in diameter were sampled with spatial coordinates in 1 cm
3
of undisturbed soil. The 55 isolates obtained yielded 42 ribotypes, covering three genomic species based on amplified ribosomal DNA restriction analysis (ARDRA) of the intergenic spacer 16S-23S, seven of which contained two to six isolates. These clonemates (identical ARDRA patterns) could be found in the same microsample or 1 cm apart. The genetic diversity did not change with distance, indicating the same habitat variability across the cube. The mixing of ribotypes, as assessed by the spatial position of clonemates, corresponded to an overlapping of clones. Although the population probably was in a recession stage in the cube (10
3
agrobacteria g
−1
), a high genetic diversity was maintained. In two independent microsamples (500 μm in diameter) at the invasion stage, the average genetic diversity was at the same level as in the cube. Quantification of the microdiversity landscape will help to estimate the probability of encounter between bacteria under realistic natural conditions and to set appropriate sampling strategies for population genetic analysis.
Solar UV-B Radiation Inhibits the Growth of Antarctic Terrestrial Fungi
Authors: Hughes Kevin A. , Lawley Blair , Newsham Kevin K.
Published: March 2003

We tested the effects of solar radiation, and UV-B in particular, on the growth of Antarctic terrestrial fungi. The growth responses to solar radiation of five fungi,Geomyces pannorum,Phoma herbarum,Pythiumsp.,Verticilliumsp., andMortierella parvispora, each isolated from Antarctic terrestrial habitats, were examined on an agar medium in the natural Antarctic environment. A 3-h exposure to solar radiation of >287 nm reduced the hyphal extension rates of all species relative to controls kept in the dark.Pythiumsp. cultures exposed to solar radiation for 1.5 h on five consecutive days were most sensitive to radiation of >287 nm, but radiation of >313 nm also inhibited growth to a lesser extent. Radiation of >400 nm had no effect on hyphal growth relative to controls kept in the dark. Short-wave solar UV-B radiation of between 287 and 305 nm inhibited the growth ofPythiumsp. hyphae on and below the surface of the agar medium after 24 h, but radiation of ≥345 nm only reduced the growth of surface hyphae. Similar detrimental effects of UV-B on surface and, to a lesser extent, submerged hyphae of all five fungi were shown in the laboratory by using artificial UV-B from fluorescent lamps. A comparison of growth responses to solar radiation and temperature showed that the species that were most resistant to UV radiation grew fastest at higher temperatures. These data suggest that solar UV-B reduces the growth of fungi on the soil surface in the Antarctic terrestrial environment.
Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase
Authors: Bergendahl Veit , Heyduk Tomasz , Burgess Richard R.
Published: March 2003

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (
Escherichia coli
) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.
Ethanol Tolerance in the YeastSaccharomyces cerevisiaeIs Dependent on Cellular Oleic Acid Content
Authors: You Kyung Man , Rosenfield Claire-Lise , Knipple Douglas C.
Published: March 2003

In this investigation, we examined the effects of different unsaturated fatty acid compositions ofSaccharomyces cerevisiaeon the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition ofS. cerevisiaeis relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ9Z-C16:1) and oleic acid (Δ9Z-C18:1), with the former predominating. Both UFAs are formed inS. cerevisiaeby the oxygen- and NADH-dependent desaturation of palmitic acid (C16:0) and stearic acid (C18:0), respectively, catalyzed by a single integral membrane desaturase encoded by theOLE1gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficientole1knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ9and Δ11) and substrate chain-length preferences (i.e., C16:0and C18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance iscis-vaccenic acid (Δ11Z-C18:1), whereas neither Δ11Z-C16:1nor palmitoleic acid (Δ9Z-C16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturaseTniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.
Development and Evaluation of PCR Assays for the Detection of
Paenibacillus larvae
in Honey Samples: Comparison with Isolation and Biochemical Characterization
Authors: Bakonyi Tamás , Derakhshifar Irmgard , Grabensteiner Elvira , Nowotny Norbert
Published: March 2003

PCR assays were developed for the direct detection of
Paenibacillus larvae
in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from
P. larvae
cultures. The specificity of the primers was assessed by analyzing related
Paenibacillus
and
Bacillus
strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for
P. larvae
spores. The most sensitive primer pair detected
P. larvae
in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not
P. larvae
, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.
Bioaugmentation as a Tool To Protect the Structure and Function of an Activated-Sludge Microbial Community against a 3-Chloroaniline Shock Load
Authors: Boon Nico , Top Eva M. , Verstraete Willy , Siciliano Steven D.
Published: March 2003

Bioaugmentation of bioreactors focuses on the removal of xenobiotics, with little attention typically paid to the recovery of disrupted reactor functions such as ammonium-nitrogen removal. Chloroanilines are widely used in industry as a precursor to a variety of products and are occasionally released into wastewater streams. This work evaluated the effects on activated-sludge reactor functions of a 3-chloroaniline (3-CA) pulse and bioaugmentation by inoculation with the 3-CA-degrading strain
Comamonas testosteroni
I2
gfp.
Changes in functions such as nitrification, carbon removal, and sludge compaction were studied in relation to the sludge community structure, in particular the nitrifying populations. Denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH) were used to characterize and enumerate the ammonia-oxidizing microbial community immediately after a 3-CA shock load. Two days after the 3-CA shock, ammonium accumulated, and the nitrification activity did not recover over a 12-day period in the nonbioaugmented reactors. In contrast, nitrification in the bioaugmented reactor started to recover on day 4. The DGGE patterns and the FISH and real-time PCR data showed that the ammonia-oxidizing microbial community of the bioaugmented reactor recovered in structure, activity, and abundance, while the number of ribosomes of the ammonia oxidizers in the nonbioaugmented reactor decreased drastically and the community composition changed and did not recover. The settleability of the activated sludge was negatively influenced by the 3-CA addition, with the sludge volume index increasing by a factor of 2.3. Two days after the 3-CA shock in the nonbioaugmented reactor, chemical oxygen demand (COD) removal efficiency decreased by 36% but recovered fully by day 4. In contrast, in the bioaugmented reactor, no decrease of the COD removal efficiency was observed. This study demonstrates that bioaugmentation of wastewater reactors to accelerate the degradation of toxic chlorinated organics such as 3-CA protected the nitrifying bacterial community, thereby allowing faster recovery from toxic shocks.
Seasonal Abundance of Total and Pathogenic
Vibrio parahaemolyticus
in Alabama Oysters
Authors: DePaola Angelo , Nordstrom Jessica L. , Bowers John C. , Wells Joy G. , Cook David W.
Published: March 2003

Recent
Vibrio parahaemolyticus
outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of
V. parahaemolyticus
were determined by using DNA probes targeting the thermolabile hemolysin (
tlh
) and thermostable direct hemolysin (
tdh
) genes for confirmation of total and pathogenic
V. parahaemolyticus
, respectively.
V. parahaemolyticus
was detected in all samples with densities ranging from <10 to 12,000 g
−1
. Higher
V. parahaemolyticus
densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992
V. parahaemolyticus
isolates from enrichments and direct plates, respectively, hybridized with the
tdh
probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a
tdh
-related hemolysin (
trh
) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with
tdh
+
V. parahaemolyticus
than previously reported.
Fnr Is Involved in Oxygen Control of
Herbaspirillum seropedicae
N-Truncated NifA Protein Activity in
Escherichia coli
Authors: Monteiro Rose A. , de Souza Emanuel M. , Yates M. Geoffrey , Pedrosa Fabio O. , Chubatsu Leda S.
Published: March 2003

Herbaspirillum seropedicae
is an endophytic diazotroph belonging to the β-subclass of the class
Proteobacteria
, which colonizes many members of the Gramineae. The activity of the NifA protein, a transcriptional activator of
nif
genes in
H. seropedicae
, is controlled by ammonium ions through its N-terminal domain and by oxygen through mechanisms that are not well understood. Here we report that the NifA protein of
H. seropedicae
is inactive and more susceptible to degradation in an
fnr Escherichia coli
background. Both effects correlate with oxygen exposure and iron deprivation. Our results suggest that the oxygen sensitivity and iron requirement for
H. seropedicae
NifA activity involve the Fnr protein.
Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading
Variovorax
Strain
Authors: Dejonghe Winnie , Berteloot Ellen , Goris Johan , Boon Nico , Crul Katrien , Maertens Siska , Höfte Monica , De Vos Paul , Verstraete Willy , Top Eva M.
Published: March 2003

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains,
Variovorax
sp. strain WDL1,
Delftia acidovorans
WDL34, and
Pseudomonas
sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e.,
N
,
O
-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively,
Hyphomicrobium sulfonivorans
WDL6 and
Comamonas testosteroni
WDL7. Of these five strains, only
Variovorax
sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains.
D. acidovorans
WDL34 and
C. testosteroni
WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and
H. sulfonivorans
WDL6 was the only strain able to degrade
N
,
O
-dimethylhydroxylamine. The role of
Pseudomonas
sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate,
Variovorax
sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.
2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in
Clostridium acetobutylicum
Authors: Watrous Mary M. , Clark Sandra , Kutty Razia , Huang Shouqin , Rudolph Frederick B. , Hughes Joseph B. , Bennett George N.
Published: March 2003

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in
Clostridium acetobutylicum
was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a

K
m

of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (
R
2
= 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in
C. acetobutylicum
in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.