bluuebunny/crossref_metadata_2025 huggingface dataset:
High Prevalence of Oxacillin-Resistant
Staphylococcus aureus
Isolates from Hospitalized Patients in Asia-Pacific and South Africa: Results from SENTRY Antimicrobial Surveillance Program, 1998-1999
Authors: Bell Jan M. , Turnidge John D.
Published: July 2002

As part of the SENTRY antimicrobial surveillance program, we examined the prevalence rates, types, and antibiograms of oxacillin-resistant
Staphylococcus aureus
from hospitalized patients from 17 institutions in eight countries in Asia-Pacific and South Africa (APAC). From April 1998 to December 1999, a total of 1,711 isolates of
S. aureus
(814 from blood, 392 from the respiratory tract, 467 from skin and skin structures, and 38 from urine) were collected from hospitalized patients within the APAC region. Multidrug-resistant oxacillin-resistant
S. aureus
(MORSA) isolates, defined as strains with three or more resistances to drug classes other than β-lactams, were the most common type of oxacillin-resistant
S. aureus
(ORSA). They were the most frequently identified pathogen in wound infections and were common in bloodstream and lower respiratory tract infections. In all contributing institutions combined, more than 45% (range, 4 to 74%) of
S. aureus
isolates were oxacillin resistant, and in six institutions, this rate exceeded 60%. MORSA accounted for 91.2% of all oxacillin-resistant isolates. Distinct resistance patterns predominated at various sites within the APAC region, suggesting the local evolution of resistant clones. Non-multidrug-resistant strains were frequent in one part of Australia. No vancomycin-intermediate strains were detected, and no strains were resistant to linezolid or quinupristin-dalfopristin. MORSA strains are a very common cause of infection in hospitalized patients in the APAC region.
Enhanced Inhibition of Orthopoxvirus Replication In Vitro by Alkoxyalkyl Esters of Cidofovir and Cyclic Cidofovir
Authors: Kern Earl R. , Hartline Caroll , Harden Emma , Keith Kathy , Rodriguez Natalie , Beadle James R. , Hostetler Karl Y.
Published: July 2002

The nucleotide phosphonates cidofovir (CDV) and cyclic cidofovir (cCDV) are potent antiviral compounds when administered parenterally but are not well absorbed orally. These compounds have been reported to have activity against orthopoxvirus replication in vitro and in animal models when administered parenterally or by aerosol. To obtain better oral activity, we synthesized a novel series of analogs of CDV and cCDV by esterification with two long-chain alkoxyalkanols, 3-hexadecyloxy-1-propanol (HDP-CDV; HDP-cCDV) or 3-octadecyloxy-1-ethanol (ODE-CDV; ODE-cCDV). Their activities were evaluated and compared with those of CDV and cCDV in human foreskin fibroblast (HFF) cells infected with vaccinia virus (VV) or cowpox virus (CV) using a plaque reduction assay. The 50% effective concentrations (EC
50
s) against VV in HFF cells for CDV and cCDV were 46.2 and 50.6 μM compared with 0.84 and 3.8 μM for HDP-CDV and HDP-cCDV, respectively. The EC
50
s for ODE-CDV and ODE-cCDV were 0.20 and 1.1 μM, respectively. The HDP analogs were 57- and 13-fold more active than the parent nucleotides, whereas the ODE analogs were 231- and 46-fold more active than the unmodified CDV and cCDV. Similar results were obtained using CV. Cytotoxicity studies indicated that although the analogs were more toxic than the parent nucleotides, the selective index was increased by 4- to 13-fold. These results indicate that the alkoxyalkyl esters of CDV and cCDV have enhanced activity in vitro and need to be evaluated for their oral absorption and efficacy in animal models.
Emergence of Group A Streptococcus Strains with Different Mechanisms of Macrolide Resistance
Authors: Bingen Edouard , Leclercq Roland , Fitoussi Frédéric , Brahimi Naïma , Malbruny Brigitte , Deforche Dominique , Cohen Robert
Published: July 2002

The mechanisms of resistance to macrolides in seven group A streptococcal (
Streptococcus pyogenes
) isolates that were the cause of pharyngitis in children who were unsuccessfully treated with azithromycin (10 mg/kg of body weight/day for 3 days) were evaluated. All posttreatment strains were found to be genetically related to the pretreatment isolates by random amplified polymorphism DNA analysis and pulsed-field gel electrophoresis. Two isolates had acquired either a
mef
(A) or an
erm
(B) gene, responsible for macrolide efflux and ribosomal modification, respectively. Three isolates displayed mutations in the gene encoding the L4 ribosomal protein that is part of the exit tunnel within the 50S subunit of the bacterial ribosome. In the two remaining posttreatment strains, the mechanisms of macrolide resistance could not be elucidated.
tcrB, a Gene Conferring Transferable Copper Resistance inEnterococcus faecium: Occurrence, Transferability, and Linkage to Macrolide and Glycopeptide Resistance
Authors: Hasman Henrik , Aarestrup Frank M.
Published: July 2002

A newly discovered gene, designatedtcrB, which is located on a conjugative plasmid conferring acquired copper resistance inEnterococcus faecium, was identified in an isolate from a pig. ThetcrBgene encodes a putative protein belonging to the CPx-type ATPase family with homology (46%) to the CopB protein fromEnterococcus hirae. ThetcrBgene was found inE. faeciumisolated from pigs (75%), broilers (34%), calves (16%), and humans (10%) but not in isolates from sheep. Resistant isolates, containing thetcrBgene, grew on brain heart infusion agar plates containing up to 28 mM CuSO4compared to only 4 mM for the susceptible isolates. Copper resistance, and therefore the presence of thetcrBgene, was strongly correlated to macrolide and glycopeptide resistance in isolates from pigs, and thetcrBgene was shown to be located on the same conjugative plasmid as the genes responsible for resistance to these two antimicrobial agents. The frequent occurrence of this new copper resistance gene in isolates from pigs, where copper sulfate is being used in large amounts as feed additive, suggests that the use of copper has selected for resistance.
Antisense Downregulation of ς
32
as a Transient Metabolic Controller in
Escherichia coli
: Effects on Yield of Active Organophosphorus Hydrolase
Authors: Srivastava Ranjan , Cha Hyung Joon , Peterson Marvin S. , Bentley William E.
Published: July 2002

Plasmids containing an antisense fragment of the ς
32
gene were constructed and introduced into
Escherichia coli
cells. Downregulation of the ς
32
-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that ς
32
sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of ς
32
mRNA. While a >10-fold increase in ς
32
protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of ς
32
. Correspondingly, antisense synthesis resulted in a decreased level of a ς
32
regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without ς
32
antisense mRNA. The total yield of OPH in the presence of ς
32
antisense was, on average, 62% of the yield without antisense. However, during ς
32
antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.
Structure and Species Composition of Mercury-Reducing Biofilms
Authors: Wagner-Döbler I. , Lünsdorf H. , Lübbehüsen T. , von Canstein H. F. , Li Y.
Published: July 2002

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of
Pseudomonas putida
Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.
Limitation of Bacterial Growth by Dissolved Organic Matter and Iron in the Southern Ocean
Authors: Church Matthew J. , Hutchins David A. , Ducklow Hugh W.
Published: July 2002

The importance of resource limitation in controlling bacterial growth in the high-nutrient, low-chlorophyll (HNLC) region of the Southern Ocean was experimentally determined during February and March 1998. Organic- and inorganic-nutrient enrichment experiments were performed between 42°S and 55°S along 141°E. Bacterial abundance, mean cell volume, and [
3
H]thymidine and [
3
H]leucine incorporation were measured during 4- to 5-day incubations. Bacterial biomass, production, and rates of growth all responded to organic enrichments in three of the four experiments. These results indicate that bacterial growth was constrained primarily by the availability of dissolved organic matter. Bacterial growth in the subtropical front, subantarctic zone, and subantarctic front responded most favorably to additions of dissolved free amino acids or glucose plus ammonium. Bacterial growth in these regions may be limited by input of both organic matter and reduced nitrogen. Unlike similar experimental results in other HNLC regions (subarctic and equatorial Pacific), growth stimulation of bacteria in the Southern Ocean resulted in significant biomass accumulation, apparently by stimulating bacterial growth in excess of removal processes. Bacterial growth was relatively unchanged by additions of iron alone; however, additions of glucose plus iron resulted in substantial increases in rates of bacterial growth and biomass accumulation. These results imply that bacterial growth efficiency and nitrogen utilization may be partly constrained by iron availability in the HNLC Southern Ocean.
A Selective Medium and a Specific Probe for Detection of
Vibrio vulnificus
Authors: Cerdà-Cuéllar Marta , Jofre Joan , Blanch Anicet R.
Published: July 2002

A selective medium (VVM) and a specific 16S rRNA gene (rDNA) probe (V3VV) for the detection of
Vibrio vulnificus
were developed. The medium contains
d
-(+)-cellobiose as the main carbon source and electrolytes (MgCl
2
-6H
2
O and KCl), which stimulate bacterial growth. Polymyxin B, colistin, and moderate alkalinity and salinity provide selectivity properties.
V. vulnificus
grows on VVM as flat, bright yellow colonies. Other
Vibrio
species tested either did not grow or showed green-bluish colonies, with the exception of
V. campbelli
,
V. carchariae
, and
V. navarrensis
. There is a higher colony count on VVM agar than on cellobiose-colistin agar or on modified cellobiose-polymyxin B-colistin agar. The specific probe was evaluated by colony hybridization and dot blot hybridization with PCR-amplified 16S rDNA using collection strains and environmental isolates. No strain studied other than
V. vulnificus
showed positive hybridization with this oligonucleotide. The combined use of VVM agar and the V3VV probe provided the recovery of
V. vulnificus
from mixed bacterial suspensions and spiked mussels.
The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN′
Authors: Taguchi Seiichi , Komada Shingo , Momose Haruo
Published: July 2002

To ascertain whether position 131 of a mesophilic protease, subtilisin BPN′, is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492–495, 1998), a full set of subtilisin BPN′ mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN′ and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (
k
cat
/

K
m

) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in
k
cat
and a decrease in

K
m

. All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.
Characterization of Plasmid-Borne and Chromosome-Encoded Traits of
Agrobacterium
Biovar 1, 2, and 3 Strains from France
Authors: Ridé Michel , Ridé Suzanne , Petit Annik , Bollet Claude , Dessaux Yves , Gardan Louis
Published: July 2002

We collected 111
Agrobacterium
isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits. Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3. A fourth phenon was identified which comprises three atypical strains. The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of
Agrobacterium
. Our results also suggest that biovar 1 and 2 represent distinct species. Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named
Agrobacterium vitis
) and of
Agrobacterium
grapevine strains in general. Among pathogenic strains of
Agrobacterium
, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine. We have named this compound ridéopine.
Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured
Escherichia coli
O157:H7 from Foods
Authors: Hara-Kudo Y. , Ikedo M. , Kodaka H. , Nakagawa H. , Goto K. , Masuda T. , Konuma H. , Kojima T. , Kumagai S.
Published: July 2002

We studied injury of
Escherichia coli
O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured
E. coli
O157:H7 cells from foods. Food samples inoculated with
E. coli
O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of
E. coli
O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect
E. coli
O157:H7 in food samples artificially contaminated with freeze-injured
E. coli
O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified
E. coli
broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating
E. coli
O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of
E. coli
O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating
E. coli
O157:H7 from frozen foods contaminated with injured
E. coli
O157:H7 cells.
Introduction of Raw Starch-Binding Domains into
Bacillus subtilis
α-Amylase by Fusion with the Starch-Binding Domain of
Bacillus
Cyclomaltodextrin Glucanotransferase
Authors: Ohdan Kohji , Kuriki Takashi , Takata Hiroki , Kaneko Hiroki , Okada Shigetaka
Published: July 2002

We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of
Bacillus subtilis
X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of
Bacillus
A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.
Common Degradative Pathways of Morpholine, Thiomorpholine, and Piperidine by
Mycobacterium aurum
MO1: Evidence from
1
H-Nuclear Magnetic Resonance and Ionspray Mass Spectrometry Performed Directly on the Incubation Medium
Authors: Combourieu Bruno , Besse Pascale , Sancelme Martine , Godin Jean-Philippe , Monteil André , Veschambre Henri , Delort Anne-Marie
Published: July 2002

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by
Mycobacterium aurum
MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing
1
H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by
M. aurum
MO1. The first step of the degradative pathway is cleavage of the C—N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus
Mycobacterium.
Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays
Authors: Small Jack , Call Douglas R. , Brockman Fred J. , Straub Timothy M. , Chandler Darrell P.
Published: July 2002

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from
Geobacter chapellei
and
Desulfovibrio desulfuricans
were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from
Geobacter
and
Desulfovibrio
were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect
G. chapellei
16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for
G. chapellei
16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 10
6
Geobacter
cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.
Copper-Induced Inhibition of Growth of
Desulfovibrio desulfuricans
G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead
Authors: Sani Rajesh Kumar , Peyton Brent M. , Brown Laura T.
Published: July 2002

The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using
Desulfovibrio desulfuricans
G20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of
D. desulfuricans
membrane integrity. The results show that
D. desulfuricans
in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.
Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters
Authors: Alexandrino Maria , Knief Claudia , Lipski André
Published: July 2002

Deuterated styrene ([2H8]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [2H8]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [2H8]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1cis11). These lipid markers indicated that styrene was degraded by organisms with aPseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid andcis-11-hexadecenoic acid (16:1cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such asXanthomonas,Bacillus,Streptomyces, orGordoniaspp.
Recovery and Phylogenetic Analysis of
nifH
Sequences from Diazotrophic Bacteria Associated with Dead Aboveground Biomass of
Spartina alterniflora
Authors: Lovell Charles R. , Friez Michael J. , Longshore John W. , Bagwell Christopher E.
Published: July 2002

DNA was extracted from dry standing dead
Spartina alterniflora
stalks as well as dry
Spartina
wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial
nifH
sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from α-
Proteobacteria
, and 4 were very similar (>99%) to that of
Azospirillum brasilense
. Seven sequences clustered with those from known γ-
Proteobacteria
and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead
Spartina
biomass in these two salt marshes were very similar, and relatively few major lineages were represented.
Sensitization of
Listeria monocytogenes
to Low pH, Organic Acids, and Osmotic Stress by Ethanol
Authors: Barker Clive , Park Simon F.
Published: July 2002

The killing of
Listeria monocytogenes
following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.
Growth and Arginine Metabolism of the Wine Lactic Acid BacteriaLactobacillus buchneriandOenococcus oeniat Different pH Values and Arginine Concentrations
Authors: Mira de Orduña Ramón , Patchett Mark L. , Liu Shao-Quan , Pilone Gordon J.
Published: July 2002

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth ofLactobacillus buchneriCUC-3 andOenococcus oenistrains MCW and Lo111 in wine were investigated. In contrast toL. buchneriCUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth ofL. buchneriCUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.
Purification, Characterization, and Gene Cloning of Purine Nucleosidase from
Ochrobactrum anthropi
Authors: Ogawa Jun , Takeda Sou , Xie Sheng-Xue , Hatanaka Haruyo , Ashikari Toshihiko , Amachi Teruo , Shimizu Sakayu
Published: July 2002

A bacterium,
Ochrobactrum anthropi
, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible
N
-riboside hydrolysis of purine nucleosides, the

K
m

values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with

K
i

and

K
i

′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn
2+
or Co
2+
restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in
Escherichia coli
. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH
2
-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.
The
celA
Gene, Encoding a Glycosyl Hydrolase Family 3 β-Glucosidase in
Azospirillum irakense,
Is Required for Optimal Growth on Cellobiosides
Authors: Faure Denis , Henrissat Bernard , Ptacek David , Bekri My Ali , Vanderleyden Jos
Published: July 2002

The CelA β-glucosidase of
Azospirillum irakense,
belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C
3
, C
4
, and C
5
oligosaccharides. The growth of a Δ
celA
mutant on these cellobiosides was affected. In
A. irakense
, the GHF3 β-glucosidases appear to be functional alternatives for the GHF1 β-glucosidases in the assimilation of β-glucosides by other bacteria.
Identification and Disruption of the
proBA
Locus in
Listeria monocytogenes
: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection
Authors: Sleator Roy D. , Gahan Cormac G. M. , Hill Colin
Published: July 2002

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the
proBA
mutant
Escherichia coli
CSH26, we identified a listerial
proBA
operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in
E. coli
. The first gene of the operon,
proB
, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of
proB
by 17 bp is
proA
, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of
proBA
had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of
Listeria monocytogenes
, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.
Laboratory-Scale Evidence for Lightning-Mediated Gene Transfer in Soil
Authors: Demanèche Sandrine , Bertolla Franck , Buret François , Nalin Renaud , Sailland Alain , Auriol Philippe , Vogel Timothy M. , Simonet Pascal
Published: July 2002

Electrical fields and current can permeabilize bacterial membranes, allowing for the penetration of naked DNA. Given that the environment is subjected to regular thunderstorms and lightning discharges that induce enormous electrical perturbations, the possibility of natural electrotransformation of bacteria was investigated. We demonstrated with soil microcosm experiments that the transformation of added bacteria could be increased locally via lightning-mediated current injection. The incorporation of three genes coding for antibiotic resistance (plasmid pBR328) into the
Escherichia coli
strain DH10B recipient previously added to soil was observed only after the soil had been subjected to laboratory-scale lightning. Laboratory-scale lightning had an electrical field gradient (700 versus 600 kV m
−1
) and current density (2.5 versus 12.6 kA m
−2
) similar to those of full-scale lightning. Controls handled identically except for not being subjected to lightning produced no detectable antibiotic-resistant clones. In addition, simulated storm cloud electrical fields (in the absence of current) did not produce detectable clones (transformation detection limit, 10
−9
). Natural electrotransformation might be a mechanism involved in bacterial evolution.
Differentiation of Strains of
Xylella fastidiosa
by a Variable Number of Tandem Repeat Analysis
Authors: Coletta-Filho Helvécio Della , Takita Marco Aurélio , de Souza Alessandra Alves , Aguilar-Vildoso Carlos Ivan , Machado Marcos Antonio
Published: July 2002

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen
Xylella fastidiosa
and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27
X. fastidiosa
strains from different hosts. The highest average Nei's measure of genetic diversity (
H
) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus
X. fastidiosa
strains, some specific VNTR loci had a
H
value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific
X. fastidiosa
strains.
DNA from Uncultured Organisms as a Source of 2,5-Diketo-
d
-Gluconic Acid Reductases
Authors: Eschenfeldt William H. , Stols Lucy , Rosenbaum Helga , Khambatta Zubin S. , Quaite-Randall Elsie , Wu Shan , Kilgore Donna C. , Trent Jonathan D. , Donnelly Mark I.
Published: July 2002

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-
d
-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in
Escherichia coli.
The purified gene products catalyzed the reduction of 2,5-diketo-
d
-gluconic acid to 2-keto-
l
-gulonic acid. Compared to previously described DKGRs isolated from
Corynebacterium
spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher
k
cat
/
K

m

values than those previously determined, primarily as a result of better binding of substrate. The
K

m

values for the two new reductases were 57 and 67 μM, versus 2 and 13 mM for the
Corynebacterium
enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.
Xylulokinase Overexpression in Two Strains ofSaccharomyces cerevisiaeAlso Expressing Xylose Reductase and Xylitol Dehydrogenase and Its Effect on Fermentation of Xylose and Lignocellulosic Hydrolysate
Authors: Johansson Björn , Christensson Camilla , Hobley Timothy , Hahn-Hägerdal Bärbel
Published: July 2002

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genesXYL1(xylose reductase) andXYL2(xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The geneXKS1encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect ofXKS1overexpression on two differentS. cerevisiaehost strains, H158 and CEN.PK, also expressingXYL1andXYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizingS. cerevisiaestrains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate.XKS1overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However,XKS1overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation ofXKS1expression are important for generating an efficient xylose-fermenting recombinant strain ofS. cerevisiae.
Transcriptional Regulation of
Nostoc
Hydrogenases: Effects of Oxygen, Hydrogen, and Nickel
Authors: Axelsson Rikard , Lindblad Peter
Published: July 2002

The transcription of structural genes encoding two hydrogenases in N
2
-fixing cultures of the cyanobacteria
Nostoc muscorum
and
Nostoc
sp. strain PCC 73102 were examined by reverse transcription-PCR. A low level of oxygen and addition of nickel induce higher transcript levels of both hydrogenases, whereas molecular hydrogen has a positive effect on the transcription of the genes encoding only the uptake hydrogenase.
Wax Ester Production from
n
-Alkanes by
Acinetobacter
sp. Strain M-1: Ultrastructure of Cellular Inclusions and Role of Acyl Coenzyme A Reductase
Authors: Ishige Takeru , Tani Akio , Takabe Keiji , Kawasaki Kazunori , Sakai Yasuyoshi , Kato Nobuo
Published: July 2002

Acinetobacter
sp. strain M-1 accumulated a large amount of wax esters from an
n
-alkane under nitrogen-limiting conditions. Under the optimized conditions with
n
-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.
Genetic Relationship among Worldwide Strains of
Xanthomonas
Causing Canker in Citrus Species and Design of New Primers for Their Identification by PCR
Authors: Cubero J. , Graham J. H.
Published: July 2002

Partial sequence analysis of the ribosomal operon in
Xanthomonas axonopodis
allowed discrimination among strains causing the A, B, and C types of citrus bacterial canker (CBC) and quantification of the relationship of these organisms with other species and pathovars in the same genus. Sets of primers based on sequence differences in the internally transcribed spacer and on a sequence from the plasmid gene
pthA
involved in virulence were designed for specific identification of xanthomonads causing CBC diseases. The two sets were validated with a collection of
Xanthomonas
strains associated with citrus species. The primer set based on ribosomal sequences had a high level of specificity for
X. axonopodis
pv. citri, whereas the set based on the
pthA
gene was universal for all types of CBC organisms. Moreover, the relationships among worldwide
Xanthomonas
strains causing CBC were analyzed by amplification of repetitive sequences (enterobacterial repetitive intergenic consensus and BOX elements). Under specific conditions, pathotypes of these
Xanthomonas
strains could be discerned, and subgroups of the pathotypes were identified. Subgroups of strains were associated with certain geographic areas of the world, and on this basis the origin of type A strains introduced into Florida could be inferred.
Reduction of Olive Knot Disease by a Bacteriocin from
Pseudomonas syringae
pv. ciccaronei
Authors: Lavermicocca Paola , Lonigro Stella Lisa , Valerio Francesca , Evidente Antonio , Visconti Angelo
Published: July 2002

A bacteriocin produced by
Pseudomonas syringae
pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of
P. syringae
subsp.
savastanoi
, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from
P. syringae
pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by
P. syringae
subsp.
savastanoi
strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10
5
and 10
8
CFU ml
−1
, respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml
−1
at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml
−1
) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from
P. syringae
pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.
Fusaric Acid-Producing Strains of
Fusarium oxysporum
Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in
Pseudomonas fluorescens
CHA0 In Vitro and in the Rhizosphere of Wheat
Authors: Notz Regina , Maurhofer Monika , Dubach Helen , Haas Dieter , Défago Geneviève
Published: July 2002

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain
Pseudomonas fluorescens
CHA0. FA production by 12
Fusarium oxysporum
strains varied substantially. We measured the effect of FA production on expression of the
phlACBDE
biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational
phlA
′-′
lacZ
fusion. Only FA-producing
F. oxysporum
strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of
phlA
repression. The repressing effect of FA on
phlA
′-′
lacZ
expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of
F. oxysporum
were tested for their influence on
phlA
expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil.
F. oxysporum
strain 798 (FA
+
) repressed
phlA
expression in CHA0 significantly, whereas strain 242 (FA
−
) did not. In the
phlF
mutant CHA638,
phlA
expression was not altered by the presence of either
F. oxysporum
strain 242 or 798.
phlA
expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits
phlA
expression in the wheat rhizosphere.