bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry
Authors: Wilcox Sheri K. , Cavey Gregory S. , Pearson James D.
Published: July 2002

Mutations in several ribosomal proteins are known to be related to antibiotic resistance. For several strains of
Escherichia coli
, the mutated protein is known but the amino acid actually altered has not been documented. Characterization of these determinants for antibiotic resistance in proteins will further the understanding of the precise mechanism of the antibiotic action as well as provide markers for resistance. Mass spectrometry can be used as a valuable tool to rapidly locate and characterize mutant proteins by using a small amount of material. We have used electrospray and matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry to map out all 56 ribosomal proteins in
E. coli
based on intact molecular masses. We used this fingerprinting approach to locate variants of ribosomal proteins displaying a change in mass. In particular we have studied proteins responsible for streptomycin, erythromycin, and spectinomycin resistance in three strains of
E. coli
, and then we characterized each mutation responsible for resistance by analyzing tryptic peptides of these proteins by using MALDI-TOF and nanoelectrospray tandem mass spectrometry. The results provided markers for antibiotic resistance and demonstrated that mass spectrometry can be used to rapidly investigate changes in individual proteins from a complex with picomole amounts of protein.
Pharmacodynamics and Bactericidal Activity of Moxifloxacin in Experimental
Escherichia coli
Meningitis
Authors: Rodriguez-Cerrato Violeta , McCoig Cynthia C. , Michelow Ian C. , Ghaffar Faryal , Jafri Hasan S. , Hardy Robert D. , Patel Chetan , Olsen Kurt , McCracken George H.
Published: July 2002

Moxifloxacin, an 8-methoxyquinolone with broad-spectrum activity in vitro, was studied in the rabbit model of
Escherichia coli
meningitis. The purposes of this study were to evaluate the bactericidal effectiveness and the pharmacodynamic profile of moxifloxacin in cerebrospinal fluid (CSF) and to compare the bactericidal activity with that of ceftriaxone and meropenem therapy. After induction of meningitis, animals were given single doses of 10, 20, and 40 mg/kg or divided-dose regimens of 5, 10, and 20 mg/kg twice, separated by 6 h. After single doses, the penetration of moxifloxacin into purulent CSF, measured as percentage of the area under the concentration-time curve (AUC) in CSF relative to the AUC in plasma, was approximately 50%. After single doses of 10, 20, and 40 mg/kg, the maximum CSF concentration (
C
max
) values were 1.8, 4.2, and 4.9 μg/ml, respectively; the AUC values (total drug) were 13.4, 25.4, and 27.1 μg/ml · h, respectively, and the half-life values (
t
½
) were 6.7, 6.6, and 4.7 h, respectively. The bacterial killing in CSF for moxifloxacin, calculated as the Δlog
10
CFU per milliliter per hour, at 3, 6, and 12 h after single doses of 10, 20, and 40 mg/kg were −5.70, −6.62, and −7.02; −7.37, −7.37, and −6.87; and −6.62, −6.62, and −6.62, respectively, whereas those of ceftriaxone and meropenem were −4.18, −5.24, and −4.43, and −3.64, −3.59, and −4.12, respectively. The CSF pharmacodynamic indices of AUC/MBC and
C
max
/MBC were interrelated (
r
= 0.81); there was less correlation with
T
> MBC (
r
= 0.74). In this model, therapy with moxifloxacin appears to be at least as effective as ceftriaxone and more effective than meropenem therapy in eradicating
E. coli
from CSF.
Experimental and Conformational Analyses of Interactions between Butenafine and Lipids
Authors: Mingeot-Leclercq Marie-Paule , Gallet Xavier , Flore Christel , Van Bambeke Françoise , Peuvot Jacques , Brasseur Robert
Published: July 2002

Butenafine (
N
-4-
tert
-butylbenzyl-
N
-methyl-1-naphtalenemethylamine hydrochloride) is an antifungal agent of the benzylamine class that has excellent therapeutic efficacy and a remarkably long duration of action when applied topically to treat various mycoses. Given the lipophilic nature of the molecule, efficacy may be related to an interaction with cell membrane phospholipids and permeabilization of the fungal cell wall. Similarly, high lipophilicity could account for the long duration of action, since fixation to lipids in cutaneous tissues might allow them to act as local depots for slow release of the drug. We have therefore used computer-assisted conformational analysis to investigate the interaction of butenafine with lipids and extended these observations with experimental studies in vitro using liposomes. Conformational analysis of mixed monolayers of phospholipids with the neutral and protonated forms of butenafine highlighted a possible interaction with both the hydrophilic and hydrophobic domains of membrane phospholipids. Studies using liposomes demonstrated that butenafine increases membrane fluidity [assessed by fluorescence polarization of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenylhexatriene] and membrane permeability (studied by release of calcein from liposomes). The results show, therefore, that butenafine readily interacts with lipids and is incorporated into membrane phospholipids. These findings may help explain the excellent antifungal efficacy and long duration of action of this drug when it is used as a topical antifungal agent in humans.
MDR1
-Mediated Drug Resistance in
Candida dubliniensis
Authors: Wirsching Stephanie , Moran Gary P. , Sullivan Derek J. , Coleman David C. , Morschhäuser Joachim
Published: July 2002

Candida dubliniensis
is a recently described opportunistic fungal pathogen that is closely related to
Candida albicans. Candida dubliniensis
readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the
CdMDR1
gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of
CdMDR1
in drug resistance in
C. dubliniensis
, we constructed an
mdr1
null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the
CdMDR1
gene. Sequential deletion of both
CdMDR1
alleles was performed by the
MPA
R
-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the
mdr1
mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that
CdMDR1
confers resistance to the structurally unrelated drugs 4-nitroquinoline-
N
-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the
mdr1
mutant. In contrast,
CdMDR1
inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of
CdMDR1
in a hypersusceptible
Saccharomyces cerevisiae
strain had previously been shown to confer resistance to these compounds. The effect of
CdMDR1
inactivation was identical to that seen in two similarly constructed
C. albicans mdr1
mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of
CaMDR1
and
CdMDR1
in clinical
C. albicans
and
C. dubliniensis
strains seems to confer the same drug resistance profile in both species.
Enterocin P Causes Potassium Ion Efflux from
Enterococcus faecium
T136 Cells
Authors: Herranz Carmen , Cintas Luis M. , Hernández Pablo E. , Moll Gert N. , Driessen Arnold J. M.
Published: July 2002

Enterocin P is a bacteriocin produced by
Enterococcus faecium
P13. We studied the mechanism of its bactericidal action using enterocin-P-sensitive
E. faecium
T136 cells. The bacteriocin is incapable of dissipating the transmembrane pH gradient. On the other hand, depending on the buffer used, enterocin P dissipates the transmembrane potential. Enterocin P efficiently elicits efflux of potassium ions, but not of intracellularly accumulated anions like phosphate and glutamate. Taken together, these data demonstrate that enterocin P forms specific, potassium ion-conducting pores in the cytoplasmic membrane of target cells.
Rapid Screening Technique for Class 1 Integrons in
Enterobacteriaceae
and Nonfermenting Gram-Negative Bacteria and Its Use in Molecular Epidemiology
Authors: Maguire Alison J. , Brown Derek F. J. , Gray James J. , Desselberger Ulrich
Published: July 2002

A screening technique for integrons in members of the family
Enterobacteriaceae
and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5′ ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.
Transposons Tn
1696
and Tn
21
and Their Integrons In4 and In2 Have Independent Origins
Authors: Partridge Sally R. , Brown Heidi J. , Stokes H. W. , Hall Ruth M.
Published: July 2002

The first 13.6 kb of the mercury and multidrug resistance transposon Tn
1696
, which includes the class 1 integron In4, has been sequenced. In4 is 8.33 kb long and contains the 5′-conserved segment (5′-CS) and 2.24 kb of the 3′-conserved segment (3′-CS) flanking four integrated cassettes. The 3′-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS
6100
flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons. This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn
21
, and other related class 1 integrons. In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism. In4 lies between the
resII
and
resI
sites of a backbone mercury resistance transposon which is >99.5% identical to Tn
5036
. Although Tn
21
and Tn
1696
are both classified as members of the Tn
21
subfamily of the Tn
3
transposon family, the backbone mercury resistance transposons are only 79 to 96% identical. Tn
21
also contains a region of about 0.7 kb not found in Tn
1696
. The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance transposons. Thus, Tn
21
and Tn
1696
have independent histories and origins. Other transposons (Tn
1403
and Tn
1412
) that include a class 1 integron also have independent origins. In all except Tn
21
, the integron is located within the
res
region of the backbone transposon.
Oxacillinase-Mediated Resistance to Cefepime and Susceptibility to Ceftazidime inPseudomonas aeruginosa
Authors: Aubert Daniel , Poirel Laurent , Chevalier Jacqueline , Leotard Sophie , Pages Jean-Marie , Nordmann Patrice
Published: July 2002

Pseudomonas aeruginosaclinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D β-lactamase, OXA-1. Theoxa-31gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected inP. aeruginosaSOF-1. Resistance to cefepime and susceptibility to ceftazidime inP. aeruginosawere conferred by OXA-1 as well.
Sputum Itraconazole Concentrations in Cystic Fibrosis Patients
Authors: Sermet-Gaudelus I. , Lesne-Hulin A. , Lenoir G. , Singlas E. , Berche P. , Hennequin C.
Published: July 2002

Itraconazole diffusion in sputum was studied in 11 cystic fibrosis patients with allergic bronchopulmonary aspergillosis. There was a high interindividual variability in sputum itraconazole concentration and sputum/serum drug concentration ratio. Three children had sputum drug concentrations before oral administration that were lower than the itraconazole MIC at which 90% of
Aspergillus fumigatus
strains were inhibited, although their serum drug concentrations were within the therapeutic range.
Activities of Gemifloxacin and Five Other Antimicrobial Agents against
Listeria monocytogenes
and Coryneform Bacteria Isolated from Clinical Samples
Authors: Martı́nez-Martı́nez Luis , Joyanes Providencia , Suárez Ana I. , Perea Evelio J.
Published: July 2002

The in vitro activities of gemifloxacin, ciprofloxacin, ampicillin, doxycycline, gentamicin, and vancomycin were evaluated against 15
Listeria monocytogenes
strains and 205 coryneform bacteria isolated from clinical samples. The percentages of strains inhibited by gemifloxacin at 0.5 μg/ml were 100% (
L. monocytogenes
), 93.3% (
Brevibacterium
spp.), 90% (
Corynebacterium minutissimum
), 42.5% (
Corynebacterium amycolatum
), 20% (
Corynebacterium striatum
), 12.5% (
Corynebacterium jeikeium
), and 10% (
Corynebacterium urealyticum
). One hundred percent of the
L. monocytogenes
strains were inhibited by 0.25 μg of gemifloxacin per ml, whereas 0% of the strains were inhibited by 0.25 μg of ciprofloxacin per ml. Vancomycin at 2 μg/ml inhibited all strains. Doxycycline and gentamicin at 4 μg/ml inhibited 94 and 49% of the strains, respectively, while ampicillin at 0.5, 2, and 8 μg/ml inhibited 24, 61, and 66% of the strains, respectively. It is concluded that gemifloxacin shows good in vitro activity against
L. monocytogenes
and coryneform bacteria except
C. jeikeium
and
C. urealyticum
.
Relationship between Chloroquine Toxicity and Iron Acquisition in
Saccharomyces cerevisiae
Authors: Emerson Lyndal R. , Nau Martin E. , Martin Rodger K. , Kyle Dennis E. , Vahey Maryanne , Wirth Dyann F.
Published: July 2002

Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used
Saccharomyces cerevisiae
as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in
S. cerevisiae
. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (
SIT1
,
ARN2
,
ARN4
, and
SMF2
). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (
FET3
and
FET4
) and yeast deficient in
SIT1
, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing.
55
FeCl
3
accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.
Susceptibilities of
Mycoplasma fermentans
and
Mycoplasma hyorhinis
to Membrane-Active Peptides and Enrofloxacin in Human Tissue Cell Cultures
Authors: Nir-Paz Ran , Prévost Marie-Christine , Nicolas Pierre , Blanchard Alain , Wróblewski Henri
Published: July 2002

Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class
Mollicutes
, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of
Mycoplasma fermentans
and
Mycoplasma hyorhinis
to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 μM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 μM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 μM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 μM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.
Change in Colony Morphology of
Candida lusitaniae
in Association with Development of Amphotericin B Resistance
Authors: McClenny Nancy B. , Fei Haihua , Baron Ellen J. , Gales Ana C. , Houston Allison , Hollis Richard J. , Pfaller Michael A.
Published: July 2002

It is not uncommon to see amphotericin B treatment failure in patients with systemic infection caused by
Candida lusitaniae
. We report a patient with stage IV ovarian carcinoma and
C. lusitaniae
sepsis whose treatment with amphotericin B failed. The initial blood isolate was susceptible to amphotericin B in vitro; however, the MIC for a blood isolate recovered 7 weeks after treatment began showed a fourfold increase. Direct subculture of two positive blood samples obtained within a week of the patient's death showed the coexistence of two distinct colony color variants on CHROMagar Candida (CAC). One variant was susceptible to amphotericin B, and one was resistant. These results emphasize the importance of repeat amphotericin B susceptibility testing for patients with persistent
C. lusitaniae
infection. The presence of colony variants on CAC may signal the emergence of amphotericin B resistance in
C. lusitaniae
and should be investigated.
Mefloquine and New Related Compounds Target the F0Complex of the F0F1H+-ATPase ofStreptococcus pneumoniae
Authors: Martín-Galiano Antonio Javier , Gorgojo Begoña , Kunin Calvin M. , de la Campa Adela G.
Published: July 2002

The activities of mefloquine (MFL) and related compounds against previously characterizedStreptococcus pneumoniaestrains carrying defined amino acid substitutions in thecsubunit of the F0F1H+-ATPase were studied. In addition, a series of MFL-resistant (Mflr) strains were isolated and characterized. A good correlation was observed between inhibition of growth and inhibition of the membrane-associated F0F1H+-ATPase activity. MFL was about 10-fold more active than optochin and about 200-fold more active than quinine in inhibiting both the growth and the ATPase activities of laboratory pneumococcal strain R6. Mutant strains were inhibited by the different compounds to different degrees, depending on their specific mutations in thecsubunit. The resistant strains studied had point mutations that changed amino acid residues in either thecsubunit or theasubunit of the F0complex. Changes in thecsubunit were located in one of the two transmembrane α helices: residues M13, G14, G20, M23, and N24 of helix 1 and residues M44, G47, V48, A49, and V57 of helix 2. Changes in theasubunit were also found in either of the transmembrane α helices, helix 5 or 6: residue L186 of helix 5 and residues W206, F209, and S214 of helix 6. These results suggest that the transmembrane helices of thecandasubunits interact and that the mutated residues are important for the structure of the F0complex and proton translocation.
In Vitro Activities of Pentamidine, Pyrimethamine, Trimethoprim, and Sulfonamides against
Aspergillus
Species
Authors: Afeltra Javier , Meis Jacques F. G. M. , Vitale Roxana G. , Mouton Johan W. , Verweij Paul E.
Published: July 2002

The susceptibilities of 70 strains of
Aspergillus
species were tested against seven different sulfa drugs and pentamidine by a microdilution method with RPMI 1640 and yeast nitrogen base media. Sulfamethoxazole, sulfadiazine, and pentamidine were active in vitro. The MICs obtained with RPMI 1640 were significantly higher than those with yeast nitrogen base. More studies are needed to further elucidate the action of these drugs.
Lentivirus Lytic Peptide 1 Perturbs both Outer and Inner Membranes of
Serratia marcescens
Authors: Phadke Shruti M. , Lazarevic Vanja , Bahr Caroline C. , Islam Kazi , Stolz Donna Beer , Watkins Simon , Tencza Sarah B. , Vogel Hans J. , Montelaro Ronald C. , Mietzner Timothy A.
Published: July 2002

Bis-lentivirus lytic protein 1 (Bis-LLP1) and polymyxin B exhibited similar killing activities against
Serratia marcescens
. By electron microscopy, bis-LLP1 interacted with the outer and cytoplasmic bacterial membranes, while polymyxin B affected only the outer membrane. The results of standard biochemical probes supported the findings of the electron microscopy studies, suggesting that these antimicrobial peptides have different mechanisms of action.
Characterization of
Cryptosporidium parvum
by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Magnuson Matthew L. , Owens James H. , Kelty Catherine A.
Published: July 2002

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed
Cryptosporidium parvum
oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for
C. parvum
were distinguishable from those obtained for
Cryptosporidium muris
. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different
C. parvum
lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers.
Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture
Authors: Annweiler Eva , Materna Arne , Safinowski Michael , Kappler Andreas , Richnow Hans H. , Michaelis Walter , Meckenstock Rainer U.
Published: July 2002

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 ± 0.003 nmol min
−1
mg of protein
−1
only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.
Application of Neural Computing Methods for Interpreting Phospholipid Fatty Acid Profiles of Natural Microbial Communities
Authors: Noble Peter A. , Almeida Jonas S. , Lovell Charles R.
Published: July 2002

The microbial community compositions of surface and subsurface marine sediments and sediments lining burrows of marine polychaetes and hemichordates from the North Inlet estuary (near Georgetown, S.C.) were analyzed by comparing ester-linked phospholipid fatty acid (PLFA) profiles with a back-propagating neural network (NN). The NNs were trained to relate PLFA inputs to sediment type outputs (e.g., surface, subsurface, and burrow lining) and worm species (e.g.,
Notomastus lobatus
,
Balanoglossus aurantiacus
, and
Branchyoasychus americana
). Sensitivity analysis was used to determine which of the 60 PLFAs significantly contributed to training the NN. The NN architecture was optimized by changing the number of hidden neurons and calculating the cross-validation error between predicted and actual outputs of training and test data. The optimal NN architecture was found to be four hidden neurons with 60-input neurons representing the 60 PLFAs, and four output neurons coding for both sediment types and worm species. Comparison of cross-validation results using NNs and linear discriminant analysis (LDA) revealed that NNs had significantly fewer incorrect classifications (2.7%) than LDA (8.4%). For the NN cross-validation, both sediment type and worm species had 3 incorrect classifications out of 112. For the LDA cross-validation, sediment type and worm species had 7 and 12 incorrect classifications out of 112, respectively. Sensitivity analysis of the trained NNs revealed that 17 fatty acids explained 50% of variability in the data set. These PLFAs were highly different among sediments and burrow types, indicating significant differences in the microbiota.
Reactivation of Insertionally Inactivated Shiga Toxin 2 Genes of
Escherichia coli
O157:H7 Caused by Nonreplicative Transposition of the Insertion Sequence
Authors: Kusumoto Masahiro , Nishiya Yoshiaki , Kawamura Yoshihisa
Published: July 2002

IS
1203
v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of
Escherichia coli
O157:H7. We analyzed the transpositional mechanism of IS
1203
v in order to investigate whether the Shiga toxin 2 genes inactivated by IS
1203
v could revert to the wild type. When the transposase activity of IS
1203
v was enhanced by artificial frameshifting, IS
1203
v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS
1203
v circle consisted of the entire IS
1203
v, but an extra 3-bp sequence (ATC) intervened between the 5′ and 3′ ends of IS
1203
v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS
1203
v. In the wild-type situation, the transposition products of IS
1203
v could be observed by PCR amplification. These results show that IS
1203
v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.
The Secondary Endosymbiotic Bacterium of the Pea AphidAcyrthosiphon pisum(Insecta: Homoptera)
Authors: Fukatsu Takema , Nikoh Naruo , Kawai Rena , Koga Ryuichi
Published: July 2002

The secondary intracellular symbiotic bacterium (S-symbiont) of the pea aphidAcyrthosiphon pisumwas investigated to determine its prevalence among strains, its phylogenetic position, its localization in the host insect, its ultrastructure, and the cytology of the endosymbiotic system. A total of 14 aphid strains were examined, and the S-symbiont was detected in 4 Japanese strains by diagnostic PCR. Two types of eubacterial 16S ribosomal DNA sequences were identified in disymbiotic strains; one of these types was obtained from the primary symbiontBuchnerasp., and the other was obtained from the S-symbiont. In situ hybridization and electron microscopy revealed that the S-symbiont was localized not only in the sheath cells but also in a novel type of cells, the secondary mycetocytes (S-mycetocytes), which have not been found previously inA. pisum. The size and shape of the S-symbiont cells were different when we compared the symbionts in the sheath cells and the symbionts in the S-mycetocytes, indicating that the S-symbiont is pleomorphic under different endosymbiotic conditions. Light microscopy, electron microscopy, and diagnostic PCR revealed unequivocally that the hemocoel is also a normal location for the S-symbiont. Occasional disordered localization of S-symbionts was also observed in adult aphids, suggesting that there has been imperfect host-symbiont coadaptation over the short history of coevolution of these organisms.
Fate of Nitrate Acquired by the Tubeworm
Riftia pachyptila
Authors: Girguis Peter R. , Lee Raymond W. , Desaulniers Nicole , Childress James J. , Pospesel Mark , Felbeck Horst , Zal Franck
Published: July 2002

The hydrothermal vent tubeworm
Riftia pachyptila
lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K. Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723–1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338–340, 1993). Here we report that at an in situ concentration of 40 μM, nitrate is acquired by
R. pachyptila
at a rate of 3.54 μmol g
−1
h
−1
, while elimination of nitrite and elimination of ammonia occur at much lower rates (0.017 and 0.21 μmol g
−1
h
−1
, respectively). We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When
R. pachyptila
tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen “sink.” Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen. Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts. Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.
Polyamine Composition and Expression of Genes Related to Polyamine Biosynthesis in an Aphid Endosymbiont,
Buchnera
Authors: Nakabachi Atsushi , Ishikawa Hajime
Published: July 2002

Polyamine composition in an aphid endosymbiotic bacterium,
Buchnera
sp., was determined by high-performance liquid chromatographic analysis. We found that
Buchnera
contained virtually only a single polyamine, spermidine. The spermidine content of
Buchnera
was considerably higher in young aphids and tended to decrease with the age of the host. Expression of
speD
and
speE
, whose gene products are key enzymes in the synthesis of spermidine, was analyzed by real-time quantitative reverse transcription-PCR. It was shown that the levels of their mRNAs fluctuated in line with the spermidine content.
Chlamydophila abortus
in a Brown Skua (
Catharacta antarctica lonnbergi
) from a Subantarctic Island
Authors: Herrmann Björn , Rahman Rubaiyat , Bergström Sven , Bonnedahl Jonas , Olsen Björn
Published: July 2002

On Bird Island, South Georgia, a new strain of
Chlamydophila abortus
was detected in one Brown skua out of 37 specimens from six different seabird species. Phylogenetic analysis of the
rnpB
and
omp1
genes indicated the strain to be more closely related to
C. abortus
than to 6BC, the type strain of
Chlamydophila psittaci
.
Starvation Alters the Apparent Half-Saturation Constant for Methane in the Type II Methanotroph
Methylocystis
Strain LR1
Authors: Dunfield Peter F. , Conrad Ralf
Published: July 2002

When cells of a type II methanotrophic bacterium (
Methylocystis
strain LR1) were starved of methane, both the
K

m
(app)

and the
V
max(app)
for methane decreased. The specific affinity (

a
o
s

) remained nearly constant. Therefore, the decreased
K

m
(app)

in starved cells was probably not an adjustment to better utilize low-methane concentrations.
Detection of Indigenous
Halobacillus
Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation
Authors: Piñar Guadalupe , Ramos Cayo , Rölleke Sabine , Schabereiter-Gurtner Claudia , Vybiral Dietmar , Lubitz Werner , Denner Ewald B. M.
Published: July 2002

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to
Halobacillus litoralis
. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown
Halobacillus
species.
Oral Toxicity of
Photorhabdus
luminescens
W14 Toxin Complexes in
Escherichia
coli
Authors: Waterfield Nicholas , Dowling Andrea , Sharma Sadhana , Daborn Phillip J. , Potter Ursula , Ffrench-Constant Richard H.
Published: July 2002

Previous attempts to express the toxin complex genes of
Photorhabdus
luminescens
W14 in
Escherichia
coli
have failed to reconstitute their oral toxicity to the model insect
Manduca
sexta
. Here we show that the combination of three genes,
tcdA
,
tcdB
, and
tccC
, is essential for oral toxicity to
M
.
sexta
when expression in
E
.
coli
is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in
E
.
coli
cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of
tcdAB
(or the homologous
tcaABC
operon) with no recombinant
tccC
homolog in a different
P
.
luminescens
strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that
P
.
luminescens
K122 carries a functional
tccC
-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both
P
.
luminescens
K122 and
E
.
coli
were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression of
tcdAB
(either with or without
tccC
) in
E
.
coli
produces visible ∼25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.
Regulation of the Packaging of
Bacillus thuringiensis
δ-Endotoxins into Inclusions
Authors: Chang Lily , Grant Rian , Aronson Arthur
Published: July 2002

During sporulation, many
Bacillus thuringiensis
subspecies synthesize several related δ-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three δ-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a
B. thuringiensis
subsp.
aizawai
cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (
cry1Ab3/cry1Ca1/cry1Da1
mRNA ratio, 4:2:1), the half-lives of the
cry1Da1
and
cry1Ab3
mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these δ-endotoxins in purified inclusions from
B. thuringiensis
subsp.
aizawai
have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three
cry
genes have the same dual overlapping promoters, but the ribosome binding sequence for the
cry1Da1
gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the δ-endotoxin composition of an inclusion.
Detection and Quantification of Methyl
tert
-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR
Authors: Hristova Krassimira R. , Lutenegger Christian M. , Scow Kate M.
Published: July 2002

The fuel oxygenate methyl
tert
-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (
C

T

) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with
Escherichia coli
cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.
Characterization of Enterocoliticin, a Phage Tail-Like Bacteriocin, and Its Effect on Pathogenic
Yersinia enterocolitica
Strains
Authors: Strauch Eckhard , Kaspar Heike , Schaudinn Christoph , Dersch Petra , Madela Kazimierz , Gewinner Christina , Hertwig Stefan , Wecke Jörg , Appel Bernd
Published: July 2002

Yersinia enterocolitica
29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of
Y. enterocolitica
belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K
+
ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.
Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium
Rhodopseudomonas palustris
Authors: Egland Paul G. , Gibson Jane , Harwood Caroline S.
Published: July 2002

We isolated a strain of
Rhodopseudomonas palustris
(RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that
R. palustris
grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide.
R. palustris
uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by
R. palustris
for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.
Novel α-Amylase That Is Highly Resistant to Chelating Reagents and Chemical Oxidants from the Alkaliphilic
Bacillus
Isolate KSM-K38
Authors: Hagihara Hiroshi , Igarashi Kazuaki , Hayashi Yasuhiro , Endo Keiji , Ikawa-Kitayama Kaori , Ozaki Katsuya , Kawai Shuji , Ito Susumu
Published: July 2002

A novel α-amylase (AmyK38) was found in cultures of an alkaliphilic
Bacillus
isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus
Bacillus
. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60°C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H
2
O
2
(1.8 M).
Impact of Biocontrol
Pseudomonas fluorescens
CHA0 and a Genetically Modified Derivative on the Diversity of Culturable Fungi in the Cucumber Rhizosphere
Authors: Girlanda M. , Perotto S. , Moenne-Loccoz Y. , Bergero R. , Lazzari A. , Defago G. , Bonfante P. , Luppi A. M.
Published: July 2002

Little is known about the effects of
Pseudomonas
biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent
Pseudomonas fluorescens
CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including
Pythium
spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera
Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium
, and
Monocillium
) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.