bluuebunny/crossref_metadata_2025 huggingface dataset:
Large Drug Resistance Virulence Plasmids of Clinical Isolates ofSalmonella entericaSerovar Choleraesuis
Authors: Chu Chishih , Chiu Cheng-Hsun , Wu Wan-Yu , Chu Chi-Hong , Liu Tsui-Ping , Ou Jonathan T.
Published: July 2002

Salmonella entericaserovar Choleraesuis generally causes systemic human salmonellosis without diarrhea, and therefore, antimicrobial treatment is essential for such patients. The drug resistance information on this organism is thus of high value. Serovar Choleraesuis usually harbors a virulence plasmid (pSCV) of 50 kb in size. Of the 16 clinical isolates identified to be serovar Choleraesuis, all except one harbored a pSCV and seven of them carried a pSCV of more than 125 kb in size. A pSCV was defined as a plasmid carryingspvCand characteristic deletions detected by PCR and by DNA-DNA hybridization (for the former criterion). The results of PCR, restriction fragment profiles, and Southern DNA-DNA hybridizations of the profiles all indicated that such larger pSCVs were derived from the 50-kb plasmid recombined with non-pSCVs found in some clinical isolates. Fifteen of the 17 strains, including a laboratory strain, were then tested for drug resistance against 16 antibiotics with E-test and the dilution method. The laboratory strain, which harbored a 50-kb pSCV and a 6-kb non-pSCV, was resistant only to sulfonamides (SUL), and its resistance gene,sulII, checked with PCR and DNA-DNA hybridization, was located on the 6-kb non-pSCV. All 14 clinical strains were resistant to multiple drugs. Of the 14, 7 were resistant to SUL, and the resistance gene was located on a plasmid. ThesulIIgene, but notblaTEM-1, was carried only on the 6-kb non-pSCV. Of the remaining six large plasmids, three of 90 kb, two of 136 kb, and one of 140 kb, the last three were pSCVs and carried the other SUL gene (sulI) and theblaTEM-1gene. The six strains were also resistant to trimethoprim-sulfamethoxazole. None of the 50-kb pSCVs carried resistance genes. These drug resistance genes on the large pSCVs were apparently also acquired through recombination.
Diversity of SHV and TEM β-Lactamases in
Klebsiella pneumoniae
: Gene Evolution in Northern Taiwan and Two Novel β-Lactamases, SHV-25 and SHV-26
Authors: Chang Feng-Yee , Siu L. K. , Fung Chang-Phone , Huang Min-Hua , Ho Monto
Published: July 2002

A total of 113 blood culture isolates of
Klebsiella pneumoniae
from 10 hospitals in northern Taiwan were studied for SHV and TEM β-lactamase production.
bla
SHV
was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of
bla
SHV-1
were found at positions T18A (ThrACC→AlaGCC), L35Q (LeuCTA→GluCAA), and M129V (MetATG→ValGTG) for SHV-25 and at position A187T (AlaGCC→ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillin-clavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV β-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum β-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the β-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV β-lactamases in Taiwan.
In Vitro Activities of 10 Antifungal Drugs against 508 Dermatophyte Strains
Authors: Fernández-Torres B. , Carrillo A. J. , Martı́n E. , Del Palacio A. , Moore M. K. , Valverde A. , Serrano M. , Guarro J.
Published: July 2002

We have tested 508 strains belonging to 24 species of dermatophytes against 10 antifungal drugs following mainly the NCCLS (M38-P) standard for filamentous fungi. However, several important factors, such as the temperature (28 versus 35°C) and time of incubation (4 to 10 days versus 21 to 74 h), have been modified. The antifungals used were itraconazole, ketoconazole, miconazole, clotrimazole, voriconazole, terbinafine, amphotericin B, fluconazole, UR-9825, and G-1. In general, with the exception of fluconazole and G-1, all antifungals were shown to be highly effective.
GES-2, a Class A β-Lactamase from
Pseudomonas aeruginosa
with Increased Hydrolysis of Imipenem
Authors: Poirel Laurent , Weldhagen Gerhard F. , Naas Thierry , De Champs Christophe , Dove Michael G. , Nordmann Patrice
Published: July 2002

Pseudomonas aeruginosa
GW-1 was isolated in 2000 in South Africa from blood cultures of a 38-year-old female who developed nosocomial pneumonia. This isolate harbored a self-transferable ca. 100-kb plasmid that conferred an expanded-spectrum cephalosporin resistance profile associated with an intermediate susceptibility to imipenem. A β-lactamase gene,
bla
GES-2
, was cloned from whole-cell DNA of
P. aeruginosa
GW-1 and expressed in
Escherichia coli
. GES-2, with a pI value of 5.8, hydrolyzed expanded-spectrum cephalosporins, and its substrate profile was extended to include imipenem compared to that of GES-1, identified previously in
Klebsiella pneumoniae
. GES-2 activity was less inhibited by clavulanic acid, tazobactam and imipenem than GES-1. The GES-2 amino acid sequence differs from that of GES-1 by a glycine-to-asparagine substitution in position 170 located in the omega loop of Ambler class A enzymes. This amino acid change may explain the extension of the substrate profile of the plasmid-encoded β-lactamase GES-2.
Accumulation of 10 Fluoroquinolones by Wild-Type or Efflux Mutant
Streptococcus pneumoniae
Authors: Piddock Laura J. V. , Johnson M. M.
Published: July 2002

A method for measuring fluoroquinolone accumulation by
Streptococcus pneumoniae
was rigorously examined. The accumulation of ciprofloxacin, clinafloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, sitafloxacin, and trovafloxacin in the presence and absence of either carbonyl cyanide
m
-chlorophenyl-hydrazone (CCCP) or reserpine was determined for two wild-type fluoroquinolone-susceptible capsulated
S. pneumoniae
strains (M3 and M4) and the noncapsulated strain R6. Two efflux mutants, R6N (which overexpresses PmrA) and a mutant of M4, M22 (no expression of PmrA), were also examined. Essentially, the fluoroquinolones fell into two groups. (i) One group consisting of ciprofloxacin, grepafloxacin, and norfloxacin accumulated to 72 to 92 ng/mg (dry weight) of cells in all strains. (ii) The remainder of the agents accumulated to 3 to 30 ng/mg (dry weight) of cells. With a decrease in hydrophobicity, there was a decrease in the concentration accumulated. With an increase in the molecular weight of the free form of each agent, there was also a decrease in the concentration accumulated. The strains differed in their responses to reserpine and CCCP. For the three fluoroquinolone-susceptible strains, only reserpine had a significant effect upon accumulation of moxifloxacin and clinafloxacin by M3 and showed no effect for the other agents and strains. For M3 and M4, CCCP enhanced the concentration of ciprofloxacin and norfloxacin accumulated, whereas for R6, the effect was only statistically significant for ofloxacin. Efflux mutant M22 accumulated less ciprofloxacin, gatifloxacin, and ofloxacin than M4 did. M22 accumulated more norfloxacin than M4 did. Reserpine and CCCP had variable effects as for the other strains. Differences in the accumulation of fluoroquinolones by R6 and R6N were highly dependent upon growth phase, and only for norfloxacin was there a significant difference between two strains.
Antibacterial Activity of Licochalcone A against Spore-Forming Bacteria
Authors: Tsukiyama Ryo-Ichi , Katsura Harumi , Tokuriki Nozomu , Kobayashi Makio
Published: July 2002

Licochalcone A was isolated from the roots of licorice,Glycyrrhiza inflata, which has various uses in the food and pharmaceutical industries; isolation was followed by extraction with ethanol and column chromatography with silica gel. In this study, the activities of licochalcone A against some food contaminant microorganisms were evaluated in vitro. The vegetative cell growth ofBacillus subtiliswas inhibited in a licochalcone A concentration-dependent manner and was completely prevented by 3 μg of licochalcone A/ml. Licochalcone A showed a high level of resistance to heating at 80 to 121°C for 15 min. Licochalcone A did not inhibit the germination of heat-treated spores ofB. subtilisinduced byl-alanine. Licochalcone A showed effects against all gram-positive bacteria tested and especially was effective against allBacillusspp. tested, with MICs of 2 to 3 μg/ml, but was not effective against gram-negative bacteria or eukaryotes at 50 μg/ml. Although the cationic antimicrobial peptides protamine and ε-poly-l-lysine resulted in the loss of antimicrobial activity in the presence of either 3% (wt/vol) NaCl or protease at 20 μg/ml, the antibacterial activity of licochalcone A was resistant to these conditions. Thus, licochalcone A could be a useful compound for the development of antibacterial agents for the preservation of foods containing high concentrations of salts and proteases, in which cationic peptides might be less effective.
In Vitro
Plasmodium falciparum
Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane
Authors: Ziegler Hanne L. , Stærk Dan , Christensen Jette , Hviid Lars , Hägerstrand Henry , Jaroszewski Jerzy W.
Published: July 2002

Lupeol, which shows in vitro inhibitory activity against
Plasmodium falciparum
3D7 strain with a 50% inhibitory concentration (IC
50
) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC
50
value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro
Plasmodium
toxicity assay.
Role of the Spore Coat Layers in
Bacillus subtilis
Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation
Authors: Riesenman Paul J. , Nicholson Wayne L.
Published: July 2002

Spores of
Bacillus subtilis
possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of
B. subtilis
(containing the
gerE36
and/or
cotE::cat
mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H
2
O
2
) and various artificial and solar UV treatments. Spores of strains carrying mutations in
gerE
and/or
cotE
were very sensitive to lysozyme and to 5% H
2
O
2
, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the
gerE36
mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the
cotE::cat
mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the
gerE36
and
cotE::cat
mutations behaved like
gerE36
mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.
Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in
Escherichia coli
Authors: Nishihara Kazuyo , Kanemori Masaaki , Yanagi Hideki , Yura Takashi
Published: July 2002

To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in
Escherichia coli
, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in
E. coli
.
Sequencing and Expression of Additional Xylanase Genes from the Hyperthermophile
Thermotoga maritima
FjSS3B.1
Authors: Reeves Rosalind A. , Gibbs Moreland D. , Morris Daniel D. , Griffiths Katherine R. , Saul David J. , Bergquist Peter L.
Published: July 2002

Two genes,
xynB
and
xynC
, coding for xylanases were isolated from
Thermotoga maritima
FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to
xynB
,
xynC
appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.
In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from
Acinetobacter johnsonii
210A
Authors: Resnick Sol M. , Zehnder Alexander J. B.
Published: July 2002

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from
Acinetobacter johnsonii
strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP

n

+ AMP → ADP + polyP

n
−1

. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in
A. johnsonii
210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of
A. johnsonii
210A cell extract, MgCl
2
, polyP

n
=35

, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from
A. johnsonii
is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.
Bacterial Growth Stimulation with Exogenous Siderophore and Synthetic
N
-Acyl Homoserine Lactone Autoinducers under Iron-Limited and Low-Nutrient Conditions
Authors: Guan Le Luo , Onuki Hiroyuki , Kamino Kei
Published: July 2002

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for
Pelagiobacter
sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 μM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM
N
-acyl-octanoylhomoserine lactone (C
8
-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C
8
-HSL; however, C
8
-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.
Influence of Salts on Virus Adsorption to Microporous Filters
Authors: Lukasik Jerzy , Scott Troy M. , Andryshak Diane , Farrah Samuel R.
Published: July 2002

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl
2
, and AlCl
3
) on the adsorption of several viruses (MS2, PRD-1, φX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).
Spatial and Temporal Variations in Chitinolytic Gene Expression and Bacterial Biomass Production during Chitin Degradation
Authors: Baty Ace M. , Eastburn Callie C. , Techkarnjanaruk Somkiet , Goodman Amanda E. , Geesey Gill G.
Published: July 2002

Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day−1were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day−1on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading geneschiAandchiB. Cells displaying high levels ofchiA-chiBexpression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population.
Dissolution of Xylose Metabolism in
Lactococcus lactis
Authors: Erlandson Karn A. , Park Joo-Heon , Wissam  , El Khal  , Kao Hsin-Hsin , Basaran Pervin , Brydges Susannah , Batt Carl A.
Published: July 2002

Xylose metabolism, a variable phenotype in strains of
Lactococcus lactis
, was studied and evidence was obtained for the accumulation of mutations that inactivate the
xyl
operon. The xylose metabolism operon (
xylRAB
) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the
xyl
locus were sequenced from
L. lactis
subsp.
lactis
B-4449 (formerly
Lactobacillus xylosus
),
L. lactis
subsp.
lactis
IO-1, and
L. lactis
subsp.
lactis
210, respectively. The two environmental isolates,
L. lactis
B-4449 and
L. lactis
IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose.
L. lactis
210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl
−
. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl
+
strains. Sequence comparisons revealed a number of point mutations in the
xylA
,
xylB
, and
xylR
genes in
L. lactis
210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in
xylB
, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of
L. lactis
unable to metabolize xylose.
Investigation of Candidate Division TM7, a Recently Recognized Major Lineage of the Domain
Bacteria
with No Known Pure-Culture Representatives
Authors: Hugenholtz Philip , Tyson Gene W. , Webb Richard I. , Wagner Ankia M. , Blackall Linda L.
Published: July 2002

A molecular approach was used to investigate a recently described candidate division of the domain
Bacteria
, TM7, currently known only from environmental 16S ribosomal DNA sequence data. A number of TM7-specific primers and probes were designed and evaluated. Fluorescence in situ hybridization (FISH) of a laboratory scale bioreactor using two independent TM7-specific probes revealed a conspicuous sheathed-filament morphotype, fortuitously enriched in the reactor. Morphologically, the filament matched the description of the Eikelboom morphotype 0041-0675 widely associated with bulking problems in activated-sludge wastewater treatment systems. Transmission electron microscopy of the bioreactor sludge demonstrated that the sheathed-filament morphotype had a typical gram-positive cell envelope ultrastructure. Therefore, TM7 is only the third bacterial lineage recognized to have gram-positive representatives. TM7-specific FISH analysis of two full-scale wastewater treatment plant sludges, including the one used to seed the laboratory scale reactor, indicated the presence of a number of morphotypes, including sheathed filaments. TM7-specific PCR clone libraries prepared from the two full-scale sludges yielded 23 novel TM7 sequences. Three subdivisions could be defined based on these data and publicly available sequences. Environmental sequence data and TM7-specific FISH analysis indicate that members of the TM7 division are present in a variety of terrestrial, aquatic, and clinical habitats. A highly atypical base substitution (
Escherichia coli
position 912; C to U) for bacterial 16S rRNAs was present in almost all TM7 sequences, suggesting that TM7 bacteria, like
Archaea
, may be streptomycin resistant at the ribosome level.
Cloning and Characterization of a Periplasmic Nuclease of
Vibrio vulnificus
and Its Role in Preventing Uptake of Foreign DNA
Authors: Wu Shi-I , Lo Shi-Kan , Shao Chung-Ping , Tsai Hsing-Wen , Hor Lien-I
Published: July 2002

We have cloned a nuclease gene,
vvn
, from
Vibrio vulnificus
, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either
V. vulnificus
or recombinant
Escherichia coli
strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in
E. coli
DH5α reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient
V. vulnificus
mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either
E. coli
or
V. vulnificus
. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of
V. vulnificus
mice.
Direct Cloning from Enrichment Cultures, a Reliable Strategy for Isolation of Complete Operons and Genes from Microbial Consortia
Authors: Entcheva Plamena , Liebl Wolfgang , Johann Andre , Hartsch Thomas , Streit Wolfgang R.
Published: July 2002

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the
Escherichia coli
biotin auxotrophic strain ATCC 33767 Δ(
bio-uvrB
). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to
bio
operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (
hut
), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (
moaABCDE
).
Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining
Authors: Grégori Gérald , Citterio Sandra , Ghiani Alessandra , Labra Massimo , Sgorbati Sergio , Brown Spencer , Denis Michel
Published: July 2002

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.
Pathogenicity of
Moraxella osloensis
, a Bacterium Associated with the Nematode
Phasmarhabditis hermaphrodita
, to the Slug
Deroceras reticulatum
Authors: Tan Li , Grewal Parwinder S.
Published: July 2002

Moraxella osloensis
, a gram-negative bacterium, is associated with
Phasmarhabditis hermaphrodita
, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug,
Deroceras reticulatum
. Infective juveniles of
P. hermaphrodita
invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that
M. osloensis
alone is pathogenic to
D. reticulatum
after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of
P. hermaphrodita
to
D. reticulatum
result from the loss of
M. osloensis
from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of
D. reticulatum
in the nematode-bacterium complex and that
P. hermaphrodita
acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by
M. osloensis
is being pursued.
Management of Indigenous Plant-Microbe Symbioses Aids Restoration of Desertified Ecosystems
Authors: Requena Natalia , Perez-Solis Estefania , Azcón-Aguilar Concepción , Jeffries Peter , Barea José-Miguel
Published: July 2002

Disturbance of natural plant communities is the first visible indication of a desertification process, but damage to physical, chemical, and biological soil properties is known to occur simultaneously. Such soil degradation limits reestablishment of the natural plant cover. In particular, desertification causes disturbance of plant-microbe symbioses which are a critical ecological factor in helping further plant growth in degraded ecosystems. Here we demonstrate, in two long-term experiments in a desertified Mediterranean ecosystem, that inoculation with indigenous arbuscular mycorrhizal fungi and with rhizobial nitrogen-fixing bacteria not only enhanced the establishment of key plant species but also increased soil fertility and quality. The dual symbiosis increased the soil nitrogen (N) content, organic matter, and hydrostable soil aggregates and enhanced N transfer from N-fixing to nonfixing species associated within the natural succession. We conclude that the introduction of target indigenous species of plants associated with a managed community of microbial symbionts is a successful biotechnological tool to aid the recovery of desertified ecosystems.
Discovery and Description of Giant Submarine Smectite Cones on the Seafloor in Eyjafjordur, Northern Iceland, and a Novel Thermal Microbial Habitat
Authors: Marteinsson Viggó Thór , Kristjánsson Jakob K. , Kristmannsdóttir Hrefna , Dahlkvist Maria , Sæmundsson Kristján , Hannington Mark , Pétursdóttir Sólveig K. , Geptner Alfred , Stoffers Peter
Published: July 2002

With the submersible JAGO and by scuba diving we discovered three remarkable geothermal cones, rising 33, 25, and 45 m from the seafloor at a depth of 65 m in Eyjafjordur, northern Iceland. The greatest geothermal activity was on the highest cone, which discharged up to 50 liters of freshwater per s at 72°C and pH 10.0. The cones were built up from precipitated smectite, formed by mixing of the hot SiO2-rich geothermal fluid with the cold Mg-rich seawater. By connecting a rubber hose to one outflow, about 240 liters of pure geothermal fluids was concentrated through a 0.2-μm-pore-size filter. Among 50 thermophilic isolates, we found members ofBacillusandThermonemaand a new unidentified low-G+C gram-positive member of theBacteriaas well as one member of theArchaea,Desulfurococcus mobilis. Analysis of small-subunit rRNA genes PCR amplified and cloned directly from environmental DNA showed that 41 out of 45Bacteriasequences belonged to members of theAquificales, whereas all of the 10Archaeasequences belonged to theKorarchaeota. The physiological characteristics of isolates from different parts of the cones indicate a completely freshwater habitat, supporting the possibility of subterranean transmittance of terrestrial organisms.
Differentiation of Environmental and Clinical Isolates of
Vibrio mimicus
from
Vibrio cholerae
by Multilocus Enzyme Electrophoresis
Authors: Vieira Verônica V. , Teixeira Luiz Fernando M. , Vicente Ana Carolina P. , Momen Hooman , Salles Carlos André
Published: July 2002

In this study, we demonstrated that analyzed strains of
Vibrio mimicus
and
Vibrio cholerae
could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between
V. mimicus
and
V. cholerae
. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.
Surface Binding of Aflatoxin B
1
by Lactic Acid Bacteria
Authors: Haskard Carolyn A. , El-Nezami Hani S. , Kankaanpää Pasi E. , Salminen Seppo , Ahokas Jorma T.
Published: July 2002

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B
1
complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B
1
remained bound. Nonviable bacteria retained the highest amount of aflatoxin B
1
.
Lactobacillus rhamnosus
strain GG (ATCC 53103) and
L. rhamnosus
strain LC-705 (DSM 7061) removed aflatoxin B
1
from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B
1
to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B
1
. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B
1
released. Binding of aflatoxin B
1
appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.
Utilization of the
rpoB
Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of
Bacillus anthracis
Authors: Qi Yuan , Patra Guy , Liang Xudong , Williams Leanne E. , Rose Sharon , Redkar Rajendra J. , DelVecchio Vito G.
Published: July 2002

The potential use of
Bacillus anthracis
as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of
B. anthracis
by taking advantage of the unique nucleotide sequence of the
B. anthracis rpoB
gene. Variable region 1 of the
rpoB
gene was sequenced from 36
Bacillus
strains, including 16
B. anthracis
strains and 20 other related bacilli, and four nucleotides specific for
B. anthracis
were identified. PCR primers were selected so that two
B. anthracis
-specific nucleotides were at their 3′ ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144
B. anthracis
strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the
rpoB
-FRET assay could be used as a new chromosomal marker for rapid detection of
B. anthracis
.
Evidence for Iron-Dependent Nitrate Respiration in the Dissimilatory Iron-Reducing Bacterium
Geobacter metallireducens
Authors: Senko John M. , Stolz John F.
Published: July 2002

The dissimilatory iron-reducing bacterium
Geobacter metallireducens
was found to require iron at a concentration in excess of 50 μM for continuous cultivation on nitrate. Growth yield (∼3-fold), cytochrome
c
content (∼7-fold), and nitrate (∼4.5-fold) and nitrite (∼70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 μM iron.
Comparison of
pmoA
PCR Primer Sets as Tools for Investigating Methanotroph Diversity in Three Danish Soils
Authors: Bourne David G. , McDonald Ian R. , Murrell J. Colin
Published: July 2002

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by
amoA
-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new
pmoA-
specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.
Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis
Authors: Bunthof Christine J. , van Schalkwijk Saskia , Meijer Wilco , Abee Tjakko , Hugenholtz Jeroen
Published: July 2002

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD
Bac
Light bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of
Lactococcus lactis
MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-
p
-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.
Quantification of
Nitrosomonas oligotropha
-Like Ammonia-Oxidizing Bacteria and
Nitrospira
spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR
Authors: Dionisi Hebe M. , Layton Alice C. , Harms Gerda , Gregory Igrid R. , Robinson Kevin G. , Sayler Gary S.
Published: July 2002

Utilizing the principle of competitive PCR, we developed two assays to enumerate
Nitrosomonas oligotropha
-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus
Nitrospira
. The specificities of two primer sets, which were designed for two target regions, the
amoA
gene and
Nitrospira
16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the
amoA
gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ± 0.0022% of the total bacterial population in a municipal WWTP.
N. oligotropha
-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell,
Nitrospira
spp. represented 0.39% ± 0.28% of the biosludge population in the municipal WWTP and 0.37% ± 0.23% of the population in the industrial WWTP. The number of
Nitrospira
sp. cells in the municipal WWTP was more than 62 times greater than the number of
N. oligotropha
-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.