bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Activity of the Ketolide ABT-773
Authors: Barry Arthur L. , Fuchs Peter C. , Brown Steven D.
Published: July 2002

The in vitro activities of ABT-773, azithromycin, erythromycin, and clindamycin were compared by testing 1,223 clinical isolates selected to represent different species and phenotypes. ABT-773 was particularly potent against staphylococci (the MIC at which 90% of the strains tested were inhibited [MIC
90
] was ≤0.06 μg/ml), including all strains that were macrolide resistant but clindamycin susceptible.
Streptococcus pneumoniae
and other streptococci were inhibited by low concentrations of ABT-773, and that included most erythromycin-resistant strains. Against
Haemophilus influenzae
, ABT-773 and azithromycin were similar in their antibacterial potency (MIC
90
, 4.0 and 2.0 μg/ml, respectively).
Hyperbaric Oxygen as an Adjunct in Zygomycosis: Randomized Controlled Trial in a Murine Model
Authors: Barratt Diana Marie , Van Meter Keith , Asmar Philip , Nolan Terry , Trahan Chris , Garcia-Covarrubias Lisardo , Metzinger Stephen E.
Published: July 2002

Zygomycosis was induced by injecting CD-1 mice with 5 mg of intraperitoneal deferoxamine and then 10
6
CFU of intravenous and intrasinus
Rhizopus arrhizus
. The addition of hyperbaric oxygen (2.0 atm absolute twice daily) to amphotericin B did not improve survival over that achieved with amphotericin B and placebo air treatments.
In Vitro Activities of a New Ketolide, ABT-773, against Multidrug-Resistant Gram-Positive Cocci
Authors: Singh Kavindra V. , Malathum Kumthorn , Murray Barbara E.
Published: July 2002

The in vitro activities of ABT-773 were evaluated against 324 strains of gram-positive bacteria, including multidrug-resistant
Staphylococcus
spp. and
Enterococcus
spp. ABT-773 had lower MIC ranges, MICs at which 50% of isolates are inhibited (MIC
50
s), and MIC
90
s than erythromycin or clindamycin for almost all isolates tested. The MICs of ABT-773 were also lower than those of quinupristin-dalfopristin (Q-D) for methicillin-susceptible
Staphylococcus aureus
,
Rhodococcus
spp., and
Streptococcus
spp., while the MICs of Q-D were lower than those of ABT-773 for methicillin-resistant
S. aureus
and
Enterococcus faecium
, including vancomycin-resistant isolates.
Resistance and Adaptation to Quinidine inSaccharomyces cerevisiae: Role ofQDR1(YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance
Authors: Nunes Patrı́cia A. , Tenreiro Sandra , Sá-Correia Isabel
Published: July 2002

As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded bySaccharomyces cerevisiaeopen reading frameYIL120wis a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus namedQDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However,QDR1gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence ofQDR1.
C-Terminal Region of
Pseudomonas aeruginosa
Outer Membrane Porin OprD Modulates Susceptibility to Meropenem
Authors: Epp Simone F. , Köhler Thilo , Plésiat Patrick , Michéa-Hamzehpour Mehri , Frey Joachim , Pechère Jean-Claude
Published: July 2002

We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of
Pseudomonas aeruginosa
. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The
oprD
genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The
oprD
gene variants and the wild-type
oprD
gene were cloned and expressed in a defined
oprD
mutant. The meropenem MICs for strains carrying the
oprD
genes from clinical isolates were four times lower than that for the strain carrying the wild-type
oprD
gene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1
oprD
gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acid
l
-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.
Multidrug-Resistant Strains of
Neisseria gonorrhoeae
in Greece
Authors: Mavroidi Angeliki , Tzouvelekis Leonidas S. , Kyriakis Kyriakos P. , Avgerinou Helen , Daniilidou Maria , Tzelepi Eva
Published: July 2002

Eighty-seven out of 575 gonococci isolated in Greece from 1991 to 1998 belonged to serovar Bropyst and exhibited resistance to penicillin, tetracycline, erythromycin, and chloramphenicol. Conventional and molecular typing showed three clusters, A, B, and C, that were associated with networks of high- frequency transmitters (cluster A with homosexuals and clusters B and C with refugees from Eastern Europe). Study of one isolate revealed mutations in the
penA, mtrR
, and
porB
genes that may explain the multidrug-resistant phenotype.
Comparison of Fractional Inhibitory Concentration Index with Response Surface Modeling for Characterization of In Vitro Interaction of Antifungals against Itraconazole-Susceptible and -ResistantAspergillus fumigatusIsolates
Authors: Te Dorsthorst D. T. A. , Verweij P. E. , Meis J. F. G. M. , Punt N. C. , Mouton J. W.
Published: July 2002

Although the fractional inhibitory concentration (FIC) index is most frequently used to define or to describe drug interactions, it has some important disadvantages when used for drugs against filamentous fungi. This includes observer bias in the determination of the MIC and no agreement on the endpoints (MIC-0, MIC-1, or MIC-2 [≥95, ≥75, and ≥50% growth inhibition, respectively]) when studying drug combinations. Furthermore, statistical analysis and comparisons are troublesome. The use of a spectrophotometric method to determine the effect of drug combinations yields quantitative data and permits the use of model fits to the whole response surface. We applied the response surface model described by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331-385, 1995) to determine the interaction coefficient alpha (ICα) using a program developed for that purpose and compared the results with FIC indices. The susceptibilities of amphotericin B (AM), itraconazole (IT), and terbinafine (TB) were tested either alone or in combination against 10 IT-susceptible (IT-S) and 5 IT-resistant (IT-R) clinical strains ofAspergillus fumigatususing a modified checkerboard microdilution method that employs the dye MTT [3-(4,5-dimethyl-2-thiazyl)2,5-diphenyl-2H-tetrazolium bromide]. Growth in each well was determined by a spectrophotometer. FIC indices were determined and ICα values were estimated for each organism strain combination, and the latter included error estimates. Depending on the MIC endpoint used, the FIC index ranged from 1.016 to 2.077 for AM-IT, from 0.544 to 1.767 for AM-TB, and from 0.656 to 0.740 for IT-TB for the IT-S strains. For the IT-R strains the FIC index ranged from 0.308 to 1.767 for AM-IT, from 0.512 to 1.646 for AM-TB, and from 0.403 to 0.497 for IT-TB. The results indicate that the degree of interaction is not only determined by the agents themselves but also by the choice of the endpoint. Estimates of the ICα values showed more consistent results. Although the absolute FIC indices were difficult to interpret, there was a good correlation with the results obtained using the ICα values. The combination of AM with either IT or TB was antagonistic in vitro, whereas the combination of IT and TB was synergistic in vitro for both IT-S and IT-R strains. The use of response surface modeling to determine the interaction of drugs against filamentous fungi is promising, and more consistent results are obtained by this method than by using FIC indices.
Interaction between Polyamines and Bacterial Outer Membranes as Investigated with Ion-Selective Electrodes
Authors: Katsu Takashi , Nakagawa Hideki , Yasuda Keiko
Published: July 2002

We analyzed the interaction between polyamines and the outer membrane of
Escherichia coli
cells using potentiometric measurements with Ca
2+
, tetraphenylphosphonium (TPP
+
), and K
+
electrodes. The Ca
2+
electrode was used to examine the ability of the polyamines to release Ca
2+
from the outer membrane. The TPP
+
electrode was used to examine the ability to permeabilize the outer membrane, since the uptake of TPP
+
was enhanced when the permeability barrier of the outer membrane was disrupted. The K
+
electrode was used to examine permeabilization in the cytoplasmic membrane by monitoring the efflux of K
+
in cytosol. Although Ca
2+
release was remarkably enhanced by increasing the number of amino groups in polyamines, no TPP
+
uptake was observed with polyamines of a simple structure, such as ethylenediamine, spermidine, and spermine. TPP
+
uptake was observed when appropriate lipophilic moieties were further attached to the polyamines with three or four amino groups, indicating that the existence of bulky moieties as well as the number of amino groups is important to induce outer membrane permeabilization. Thus, 1-naphthylacetylspermine and
N,N
′-bis[6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine (methoctramine) were especially effective in increasing the permeability of the outer membrane of
E. coli
cells, being comparable to polymyxin B nonapeptide, a well-known cationic peptide showing such action.
Origin and Evolution of the AmpC β-Lactamases ofCitrobacter freundii
Authors: Barlow Miriam , Hall Barry G.
Published: July 2002

To determine whether the widespread clinical use of β-lactams has been selective forCitrobacter freundii-derived alleles of plasmidampCgenes, we generated a Bayesian consensus phylogeny of the publishedampCsequences and compared the MICs of 16 β-lactam antibiotics forEscherichia colistrains containing cloned copies of theC. freundii ampCalleles. We found that for the majority of compounds investigated, there has been essentially no increase in β-lactam resistance conferred by those alleles. We also found thatampCalleles from the chromosomes of two β-lactam-sensitiveC. freundiistrains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing β-lactam resistance inE. colias were the plasmid-borne alleles from β-lactam-resistant clinical isolates. These results suggest that selection for increased resistance to β-lactam antibiotics has not been a significant force directing the evolution of theC. freundii ampCalleles found in β-lactam-resistant clinical isolates.
Widespread Distribution of a Tet W Determinant among Tetracycline-Resistant Isolates of the Animal PathogenArcanobacterium pyogenes
Authors: Billington Stephen J. , Songer J. Glenn , Jost B. Helen
Published: July 2002

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogenArcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates ofA. pyogeneswas highly similar at the DNA level (92% identity) to thetet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacteriumButyrivibrio fibrisolvens. Thetet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, thetet(W) gene was associated with amobgene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer oftet(W) was detected frommob+A. pyogenesisolates to a tetracycline-sensitiveA. pyogenesrecipient. The mobile nature of this determinant and the presence ofA. pyogenesin the gastrointestinal tract of cattle and pigs suggest thatA. pyogenesmay have inherited this determinant within the gastrointestinal tracts of these animals.
The Presence of Humic Substances and DNA in RNA Extracts Affects Hybridization Results
Authors: Alm Elizabeth Wheeler , Zheng Dandan , Raskin Lutgarde
Published: July 2002

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.
Succession of Pelagic Marine Bacteria during Enrichment: a Close Look at Cultivation-Induced Shifts
Authors: Eilers Heike , Pernthaler Jakob , Amann Rudolf
Published: July 2002

Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S−) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction.
Vibrio
spp. and members of the
Alteromonas/Colwellia
cluster (A/C) were selectively enriched in S+ and S−, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant γ-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus
Oceanospirillum
were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of
Vibrio
and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas
Oceanospirillum
spp. did not. The life strategy of rapidly enriched culturable γ-proteobacteria could thus be described as a “feast and famine” existence involving different activation levels of substrate concentration.
Integrative Food-Grade Expression System Based on the Lactose Regulon of
Lactobacillus casei
Authors: Gosalbes María José , Esteban Carlos David , Galán José Luis , Pérez-Martínez Gaspar
Published: July 2002

The lactose operon from
Lactobacillus casei
is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of
L. casei
. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3′ end of
lacG
and the complete
lacF
gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of
L. casei
could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the
E. coli gusA
gene and the
L. lactis ilvBN
genes in
L. casei
. Following the same expression pattern as that for the lactose genes, β-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in
L. casei
that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.
PCR Bias in Ecological Analysis: a Case Study for Quantitative
Taq
Nuclease Assays in Analyses of Microbial Communities
Authors: Becker Sven , Böger Peter , Oehlmann Ralfh , Ernst Anneliese
Published: July 2002

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of
Taq
nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10
4
-fold excess of phylogenetically similar DNA and of an up to 10
7
-fold excess of dissimilar DNA.
Bacterial Biodegradation of Extractives and Patterns of Bordered Pit Membrane Attack in Pine Wood
Authors: Burnes Todd A. , Blanchette Robert A. , Farrell Roberta L.
Published: July 2002

Wood extractives, commonly referred to as pitch, cause major problems in the manufacturing of pulp and paper. Treatment of nonsterile southern yellow pine chips for 14 days with
Pseudomonas fluorescens
,
Pseudomonas
sp.,
Xanthomonas campestris
, and
Serratia marcescens
reduced wood extractives by as much as 40%. Control treatments receiving only water lost 11% of extractives due to the growth of naturally occurring microorganisms. Control treatments were visually discolored after the 14-day incubation, whereas bacterium-treated wood chips were free of dark staining. Investigations using
P. fluorescens
NRRL B21432 showed that all individual resin and fatty acid components of the pine wood extractives were substantially reduced. Micromorphological observations showed that bacteria were able to colonize resin canals, ray parenchyma cells, and tracheids. Tracheid pit membranes within bordered pit chambers were degraded after treatment with
P. fluorescens
NRRL B21432.
P. fluorescens
and the other bacteria tested appear to have the potential for biological processing to substantially reduce wood extractives in pine wood chips prior to the paper making process so that problems associated with pitch in pulp mills can be controlled.
Efficient Improvement of Silage Additives by Using Genetic Algorithms
Authors: Davies Zoe S. , Gilbert Richard J. , Merry Roger J. , Kell Douglas B. , Theodorou Michael K. , Griffith Gareth W.
Published: July 2002

The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e., no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a “fitness” value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a “cost” element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.
Molecular Identification of Species from the
Penicillium roqueforti
Group Associated with Spoiled Animal Feed
Authors: Boysen Marianne E. , Jacobsson Karl-Gustav , Schnürer Johan
Published: July 2002

The
Penicillium roqueforti
group has recently been split into three species,
P. roqueforti
,
Penicillium carneum
, and
Penicillium paneum
, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as
P. roqueforti
, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were
P. roqueforti
, 2 were
P. paneum
, and 2 were
Penicillium expansum
. No
P. carneum
isolates were found. The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records.
P. roqueforti
appears to dominate in silage, but the ecological and toxicological importance of
P. carneum
and
P. paneum
as feed spoilage fungi is not clear. This is the first report of
P. expansum
in silage.
Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific
Streptomyces
DNA Probes
Authors: Roberts Mark A. , Crawford Don L.
Published: July 2002

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36
Streptomyces
strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for
Streptomyces
, and a strain-specific probe for the biological control agent
Streptomyces lydicus
WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the
S. lydicus
WYEC108 in DNA isolated from soil.
Functional Exoenzymes as Indicators of Metabolically Active Bacteria in 124,000-Year-Old Sapropel Layers of the Eastern Mediterranean Sea
Authors: Coolen Marco J. L. , Overmann Jörg
Published: July 2002

Hydrolytic exoenzymes as indicators of metabolically active bacteria were investigated in four consecutive sapropel layers collected from bathyal sediments of the eastern Mediterranean Sea. For comparison, the organic carbon-poor layers between the sapropels, sediment from the anoxic Urania basin, and sediments of intertidal mud flats of the German Wadden Sea were also analyzed. The sapropel layers contained up to 1.5 · 10
8
bacterial cells cm
−3
, whereas cell numbers in the intermediate layers were lower by a factor of 10. In sapropels, the determination of exoenzyme activity with fluorescently labeled substrate analogues was impaired by the strong adsorption of up to 97% of the enzymatically liberated fluorophores (4-methylumbelliferone [MUF] and 7-amino-4-methylcoumarin [MCA]) to the sediment particles. Because all established methods for the extraction of adsorbed fluorophores proved to be inadequate for sapropel sediments, we introduce a correction method which is based on the measurement of equilibrium adsorption isotherms for both compounds. Using this new approach, high activities of aminopeptidase and alkaline phosphatase were detected even in a 124,000-year-old sapropel layer, whereas the activity of β-glucosidase was low in all layers. So far, it had been assumed that the organic matter which constitutes the sapropels is highly refractory. The high potential activities of bacterial exoenzymes indicate that bacteria in Mediterranean sapropels are metabolically active and utilize part of the subfossil kerogen. Since a high adsorption capacity was determined not only for the low-molecular-weight compounds MUF and MCA but also for DNA, the extraordinarily strong adsorption of structurally different substrates to the sapropel matrix appears to be the major reason for the long-term preservation of biodegradable carbon in this environment.
Energetics of Syntrophic Propionate Oxidation in Defined Batch and Chemostat Cocultures
Authors: Scholten Johannes C. M. , Conrad Ralf
Published: July 2002

Propionate consumption was studied in syntrophic batch and chemostat cocultures of
Syntrophobacter fumaroxidans
and
Methanospirillum hungatei
. The Gibbs free energy available for the H
2
-consuming methanogens was <−20 kJ mol of CH
4
−1
and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >−10 kJ mol of propionate
−1
. Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0.07 day
−1
and produced maximum biomass yields of 2.6 g mol of propionate
−1
and 7.6 g mol of CH
4
−1
for
S. fumaroxidans
and
M. hungatei
, respectively. The energy efficiency for syntrophic growth of
S. fumaroxidans
, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP
−1
. However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown
S. fumaroxidans
was surprisingly low (0.14 kJ h
−1
mol of biomass C
−1
) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.
Penetration of the Coral-Bleaching Bacterium
Vibrio shiloi
into
Oculina patagonica
Authors: Banin E. , Israely T. , Kushmaro A. , Loya Y. , Orr E. , Rosenberg E.
Published: July 2002

Inoculation of the coral-bleaching bacterium
Vibrio shiloi
into seawater containing its host
Oculina patagonica
led to adhesion of the bacteria to the coral surface via a β-
d
-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized
V. shiloi
cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-
V. shiloi
antibodies to stain the intracellular bacteria. At 29°C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of
V. shiloi
total counts (using the anti-
V. shiloi
antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular
V. shiloi
organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of
V. shiloi
toxin P activity in the coral tissue. Internalization and multiplication of
V. shiloi
are discussed in terms of the mechanism of bacterial bleaching of corals.
Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge
Authors: Imachi Hiroyuki , Sekiguchi Yuji , Kamagata Yoichi , Ohashi Akiyoshi , Harada Hideki
Published: July 2002

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with
Methanobacterium thermoautotrophicum
. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with
M. thermoautotrophicum
. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus
Desulfotomaculum
, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.
Characterization of
Leuconostoc gasicomitatum
sp. nov., Associated with Spoiled Raw Tomato-Marinated Broiler Meat Strips Packaged under Modified-Atmosphere Conditions
Authors: Björkroth K. Johanna , Geisen Rolf , Schillinger Ulrich , Weiss Norbert , De Vos Paul , Holzapfel Wilhelm H. , Korkeala Hannu J. , Vandamme Peter
Published: July 2002

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a
Leuconostoc
species resembling
Leuconostoc gelidum
caused the spoilage of the product.
Lactobacillus sakei
,
Lactobacillus curvatus
, and a gram-positive rod phenotypically similar to heterofermentative
Lactobacillus
species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called “protein swell.” This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant
Leuconostoc
species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that
L. gelidum
was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the
L. gelidum
type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the
L. gelidum
type strain. These results warrant a separate species status, and we propose the name
Leuconostoc gasicomitatum
sp. nov. for this spoilage-associated
Leuconostoc
species.
Antagonistic Interactions among Marine Pelagic Bacteria
Authors: Long Richard A. , Azam Farooq
Published: July 2002

Recent studies suggest that bacterial abundance and species diversity in the ocean's water column are variable at the millimeter scale, apparently in response to the small-scale heterogeneity in the distribution of organic matter. We hypothesized that bacterium-bacterium antagonistic interactions may contribute to variations in community structure at the microscale. We examined each of the 86 isolates for their inhibition of growth of the remaining 85 isolates by the Burkholder agar diffusion assay. More than one-half of the isolates expressed antagonistic activity, and this trait was more common with particle-associated bacteria than with free-living bacteria. This was exemplified by members of the α subclass of the class
Proteobacteria
(α-proteobacteria), in which production of antagonistic molecules was dominated by attached bacteria. We found that γ-proteobacteria (members of the orders
Alteromonadales
and
Vibrionales
) are the most prolific producers of inhibitory materials and also the most resilient to them, while members of the
Bacteriodetes
were the organisms that were least productive and most sensitive to antagonistic interactions. Widespread interspecies growth inhibition is consistent with the role of this phenomenon in structuring bacterial communities at the microscale. Furthermore, our results suggest that bacteria from pelagic marine particles may be an underutilized source of novel antibiotics.
Two Distinct Monooxygenases for Alkane Oxidation in
Nocardioides
sp. Strain CF8
Authors: Hamamura Natsuko , Yeager Chris M. , Arp Daniel J.
Published: July 2002

Alkane monooxygenases in
Nocardioides
sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C
2
to C
16
. Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from
Pseudomonas oleovorans
were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C
6
. These results indicate the presence of two distinct monooxygenases for alkane oxidation in
Nocardioides
sp. strain CF8.
Quantification of Soil-to-Plant Transport of Recombinant Nucleopolyhedrovirus: Effects of Soil Type and Moisture, Air Currents, and Precipitation
Authors: Fuxa James R. , Richter Arthur R.
Published: July 2002

Significantly more occlusion bodies (OB) of DuPont viral construct HzSNPV-LqhIT2, expressing a scorpion toxin, were transported by artificial rainfall to cotton plants from sandy soil (70:15:15 sand-silt-clay) than from silt (15:70:15) and significantly more from silt than from clay (15:15:70). The amounts transported by 5 versus 50 mm of precipitation were the same, and transport was zero when there was no precipitation. In treatments that included precipitation, the mean number of viable OB transported to entire, 25- to 35-cm-tall cotton plants ranged from 56 (clay soil, 5 mm of rain) to 226 (sandy soil, 50 mm of rain) OB/plant. In a second experiment, viral transport increased with increasing wind velocity (0, 16, and 31 km/h) and was greater in dry (−1.0 bar of matric potential) than in moist (−0.5 bar) soil. Wind transport was greater for virus in a clay soil than in silt or sand. Only 3.3 × 10
−7
(clay soil, 5 mm rain) to 1.3 × 10
−6
(sandy soil, 50 mm rain) of the OB in surrounding soil in experiment 1 or 1.1 × 10
−7
(−0.5 bar sandy soil, 16-km/h wind) to 1.3 × 10
−6
(−1.0 bar clay soil, 31-km/h wind) in experiment 2 were transported by rainfall or wind to cotton plants. This reduces the risk of environmental release of a recombinant nucleopolyhedrovirus (NPV), because only a very small proportion of recombinant virus in the soil reservoir is transported to vegetation, where it can be ingested by and replicate in new host insects.
A Novel Regulatory Gene,
Tri10
, Controls Trichothecene Toxin Production and Gene Expression
Authors: Tag Andrew G. , Garifullina Gulnara F. , Peplow Andrew W. , Ake Charles , Phillips T. D. , Hohn Thomas M. , Beremand Marian N.
Published: July 2002

We report here the characterization of
Tri10
, a novel regulatory gene within the trichothecene gene cluster. Comparison of
Tri10
genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of
Tri10
in
Fusarium sporotrichioides
abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (
Tri4
,
Tri5
,
Tri6
, and
Tri101
) and an apparent farnesyl pyrophosphate synthetase (
Fpps
) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and
Fpps
. Further analysis revealed that disruption of
Tri10
, and to a greater extent the disruption of
Tri6,
increased sensitivity to T-2 toxin under certain growth conditions. Although
Tri10
is conserved in
Fusarium graminearum
and
Fusarium sambucinum
and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which
Tri10
acts upstream of the cluster-encoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where
Tri6
is not required for the transcription of
Tri10
but is required to limit the expression of
Tri10.
Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities
Authors: Hernández-Crespo Pedro , Sait Steven M. , Hails Rosemary S. , Cory Jenny S.
Published: July 2002

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type
Autographa californica
nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host,
Mamestra brassicae
, but the recombinant was more pathogenic than the wild-type virus in the susceptible species,
Trichoplusia ni
. Both viruses took longer to kill the larvae of
M. brassicae
than to kill those of
T. ni
. However, whereas the larvae of
T. ni
were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of
M. brassicae
. Both viruses produced a greater yield in
M. brassicae
, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in
M. brassicae
, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for
M. brassicae
but increased for
T. ni
. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.
Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by
Rhodococcus erythropolis
D-1
Authors: Matsubara Toshiyuki , Ohshiro Takashi , Nishina Yoshihiro , Izumi Yoshikazu
Published: July 2002

The dibenzothiophene (DBT)-desulfurizing bacterium,
Rhodococcus erythropolis
D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from
R. erythropolis
D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the

K
m

values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of
R. erythropolis
IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using
dszD
of
R. erythropolis
IGTS8, and the enzyme was overexpressed in
Escherichia coli
. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.