bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium Lauryl Sulfate Abrogates Human Immunodeficiency Virus Infectivity by Affecting Viral Attachment
Authors: Bestman-Smith Julie , Piret Jocelyne , Désormeaux André , Tremblay Michel J. , Omar Rabeea F. , Bergeron Michel G.
Published: July 2002

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1NL4-3with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 μM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 μM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.
Pretreatment of Mice with Clindamycin Improves Survival of Endotoxic Shock by Modulating the Release of Inflammatory Cytokines
Authors: Hirata Norio , Hiramatsu Kazufumi , Kishi Kenji , Yamasaki Tohru , Ichimiya Tomoku , Nasu Masaru
Published: July 2002

Suppression of endotoxin release and subsequent production of inflammatory cytokines is crucial in the treatment of septic shock. We investigated the effect of clindamycin (CLI) on endotoxic shock induced in mice byEscherichia colilipopolysaccharide (LPS). Mice were treated with CLI (160 to 600 mg/kg) or saline and then injected withE. coliLPS andd-(+)-galactosamine intraperitoneally 0.5 h after CLI administration. Pretreatment with CLI significantly improved survival in a dose-dependent manner (CLI, at 160, 300, and 440 mg/kg) and significantly lowered the peak concentrations of tumor necrosis factor alpha and interleukin-1β(IL-1β) in serum. However, the peak concentrations of IL-6 in the sera of CLI-treated mice were higher than in control mice. Our findings suggest that CLI alters LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model.
Evaluation of a Cyclic GMP-Dependent Protein Kinase Inhibitor in Treatment of Murine Toxoplasmosis: Gamma Interferon Is Required for Efficacy
Authors: Nare Bakela , Allocco John J. , Liberator Paul A. , Donald Robert G. K.
Published: July 2002

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) is a potent inhibitor of cyclic GMP-dependent protein kinases from Apicomplexan protozoa and displays cytostatic activity againstToxoplasma gondiiin vitro.Compound 1 has now been evaluated againstT. gondiiinfections in the mouse and appeared to protect the animals when given intraperitoneally at 50 mg/kg twice daily for 10 days. However, samples from brain, spleen, and lung taken from infected treated mice revealed the presence of parasites after cessation of administration of compound 1, indicating that a transient asymptomatic parasite recrudescence occurs in all survivors. The ability of mice to controlToxoplasmainfection after compound 1 treatment has been terminated suggested that the mouse immune system plays a synergistic role with chemotherapy in controlling the infection. To explore this possibility, gamma interferon (IFN-γ)-knockout mice were infected with parasites and treated with compound 1, and survival was compared to that of normal mice. IFN-γ-knockout mice were protected againstT. gondiithroughout the treatment phase but died during the posttreatment phase in which peak recrudescence was observed in treated immunocompetent mice. These data suggest that an IFN-γ-dependent immune response was essential for controlling and resolving parasite recrudescence in mice treated with compound 1. In addition, when compound 1-cured immunocompetent mice were rechallenged with a lethal doseof T. gondii,all survived (n= 32). It appears that the cytostatic nature of compound 1 provides an “immunization” phase during chemotherapy which allows the mice to survive the recrudescence and any subsequent challenge with a lethal dose ofT. gondii.
An
Enterococcus faecalis
ABC Homologue (Lsa) Is Required for the Resistance of This Species to Clindamycin and Quinupristin-Dalfopristin
Authors: Singh Kavindra V. , Weinstock George M. , Murray Barbara E.
Published: July 2002

Enterococcus faecalis
isolates are resistant to clindamycin (CLI) and quinupristin-dalfopristin (Q-D), and this is thought to be a species characteristic. Disruption of a gene (
abc-23
, now designated
lsa
, for “lincosamide and streptogramin A resistance”) of
E. faecalis
was associated with a ≥40-fold decrease in MICs of Q-D (to 0.75 μg/ml), CLI (to 0.12 to 0.5 μg/ml), and dalfopristin (DAL) (to 4 to 8 μg/ml) for the wild-type
E. faecalis
parental strain (Q-D MIC, 32 μg/ml; CLI MIC, 32 to 48 μg/ml; DAL MIC, 512 μg/ml). Complementation of the disruption mutant with
lsa
on a shuttle plasmid resulted in restoration of the MICs of CLI, Q-D, and DAL to wild-type levels. Under high-stringency conditions,
lsa
was found in 180 of 180 isolates of
E. faecalis
but in none of 189 other enterococci. Among 19
erm
(B)-lacking
Enterococcus faecium
strains, 9 (47%) were highly susceptible to CLI (MIC, 0.06 to 0.25 μg/ml) and had DAL MICs of 4 to 16 μg/ml; for the remaining
erm
(B)-lacking
E. faecium
strains, the CLI and DAL MICs were 4 to >256 and 2 to >128 μg/ml, respectively. In contrast, none of 32
erm
(B)-lacking
E. faecalis
strains were susceptible (CLI MIC range, 16 to 32 μg/ml; DAL MIC range, ≥32 μg/ml). When
lsa
was introduced into an
E. faecium
strain initially susceptible to CLI, the MICs of CLI and DAL increased ≥60-fold and that of Q-D increased 6-fold (to 3 to 6 μg/ml). Introduction of
lsa
into two DAL-resistant (MICs, >128 μg/ml), Q-D-susceptible (MICs, 0.5 and 1.5 μg/ml)
E. faecium
strains (CLI MICs, 12 and >256 μg/ml) resulted in an increase in the Q-D MICs from 3- to 10-fold (to 8 and >32 μg/ml), respectively. Although efflux was not studied, the similarity (41 to 64%) of the predicted Lsa protein to ABC proteins such as Vga(A), Vga(B), and Msr(A) of
Staphylococcus aureus
and YjcA of
Lactococcus lactis
and the presence of Walker A and B ATP-binding motifs suggest that this resistance may be related to efflux of these antibiotics. In conclusion,
lsa
appears to be an intrinsic gene of
E. faecalis
that explains the characteristic resistance of this species to CLI and Q-D.
Nonradiolabeling Assay for WaaP, an Essential Sugar Kinase Involved in Biosynthesis of Core Lipopolysaccharide of
Pseudomonas aeruginosa
Authors: Zhao Xin , Wenzel Cory Q. , Lam Joseph S.
Published: July 2002

waaP
is present in the lipopolysaccharide (LPS) core gene clusters of a wide range of gram-negative bacteria, and is an essential gene in
Pseudomonas aeruginosa
. The WaaP protein is a sugar kinase that adds phosphate to heptose I in the core oligosaccharide. This study describes the standardization and utility of a chemiluminescence-based enzyme-linked immunosorbent assay for the detection of WaaP kinase activity. Important features of the assay include high sensitivity, the preparation of dephosphorylated LPS as a substrate, and the use of monoclonal antibody 7-4 that specifically recognizes phosphate substituents in the LPS core.
Effect of Linoleic Acid Concentration on Conjugated Linoleic Acid Production by
Butyrivibrio fibrisolvens
A38
Authors: Kim Young Jun , Liu Rui Hai , Bond Daniel R. , Russell James B.
Published: July 2002

Butyrivibrio fibrisolvens
A38 inocula were inhibited by as little as 15 μM linoleic acid (LA), but growing cultures tolerated 10-fold more LA before growth was inhibited. Growing cultures did not produce significant amounts of
cis
-9,
trans
-11 conjugated linoleic acid (CLA) until the LA concentration was high enough to inhibit biohydrogenation, growth was inhibited, and lysis was enhanced. Washed-cell suspensions that were incubated anaerobically with 350 μM LA converted most of the LA to hydrogenated products, and little CLA was detected. When the washed-cell suspensions were incubated aerobically, biohydrogenation was inhibited, CLA production was at least twofold greater, and CLA persisted. The LA isomerase reaction was very rapid, but the LA isomerase did not recycle like a normal enzyme to catalyze more substrate. Cells that were preincubated with CLA lost their ability to produce more CLA from LA, and the CLA accumulation was directly proportional (
r
2
= 0.98) to the initial cell density. Growing cells were as sensitive to CLA as LA, the LA isomerase and reductases of biohydrogenation were linked, and free CLA was not released. Because growing cultures of
B. fibrisolvens
A38 did not produce significant amounts of CLA until the LA concentration was high, biohydrogenation was arrested, and the cell density had declined, the flow of CLA from the rumen may be due to LA-dependent bacterial inactivation, death, or lysis.
Detection on Surfaces and in Caco-2 Cells of
Campylobacter jejuni
Cells Transformed with New
gfp, yfp
, and
cfp
Marker Plasmids
Authors: Miller William G. , Bates Anne H. , Horn Sharon T. , Brandl Maria T. , Wachtel Marian R. , Mandrell Robert E.
Published: July 2002

We have developed two sets of
Campylobacter
shuttle vectors containing either the
gfp
(green fluorescent protein),
yfp
(yellow fluorescent protein), or
cfp
(cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus
Campylobacter
promoter sequence (P
c
). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments.
C. jejuni
cells transformed with the P
c
fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each
C. jejuni
strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many
C. jejuni
strains, >91% of the cells transformed with the P
c
fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent
C. jejuni
transformants suggest that aggregates containing two or more strains of
C. jejuni
may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of
C. jejuni
, by selecting single colonies, from an environmental sample may not always yield a single strain.
Molecular and Phenotypic Characterization of
Pseudomonas
spp. Isolated from Milk
Authors: Wiedmann Martin , Weilmeier Denise , Dineen Sean S. , Ralyea Robert , Boor Kathryn J.
Published: July 2002

Putative
Pseudomonas
spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species
P. putida
(cluster A),
P. fluorescens
(cluster B),
P. fragi
(as identified by Biolog) or
P. fluorescens
(as identified by API 20 NE) (cluster C),
P. fragi
(as identified by Biolog) or
P. putida
(as identified by API 20 NE) (cluster D), and
P. fluorescens
(cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy
Pseudomonas
isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for
Pseudomonas
spp.
Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis
Authors: Dunbar John , Ticknor Lawrence O. , Kuske Cheryl R.
Published: July 2002

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662–1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from
Rsa
I,
Hha
I,
Hae
III, and
Msp
I profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.
Physiological and Chemical Investigations into Microbial Degradation of Synthetic Poly(
cis
-1,4-isoprene)
Authors: Bode Helge B. , Zeeck Axel , Plückhahn Kirsten , Jendrossek Dieter
Published: July 2002

Streptomyces coelicolor
1A and
Pseudomonas citronellolis
were able to degrade synthetic high-molecular-weight poly(
cis
-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain
Streptomyces lividans
1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for
S. coelicolor
1A and
P. citronellolis
, whereas the molecular weight distribution for the control (
S. lividans
1326) remained almost constant. Three degradation products were isolated from the culture fluid of
S. coelicolor
1A grown on vulcanized rubber and were identified as (
6Z
)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (
5Z
)-6-methyl-undec-5-ene-2,9-dione, and (
5Z
,
9Z
)-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(
cis
-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of β-oxidation, (iii) oxidation of the conjugated double bond resulting in a β-keto acid, and (iv) decarboxylation.
Microbial Activity in Aquatic Environments Measured by Dimethyl Sulfoxide Reduction and Intercomparison with Commonly Used Methods
Authors: Griebler Christian , Slezak Doris
Published: July 2002

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN3, KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 × 10−17± 0.12 × 10−17mol of DMS per produced cell (mean ± standard error;R2= 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (theR2values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (theR2values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity.
Isolation and Characterization of Five
Erwinia amylovora
Bacteriophages and Assessment of Phage Resistance in Strains of
Erwinia amylovora
Authors: Schnabel Elise L. , Jones Alan L.
Published: July 2002

Phages able to infect the fire blight pathogen
Erwinia amylovora
were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages φEa1 and φEa7 and 3 novel phages named φEa100, φEa125, and φEa116C, were identified based on differences in genome size and restriction fragment pattern. φEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages φEa100, φEa7, and φEa125 each had genomes of approximately 35 kb and could be distinguished by their
Eco
RI restriction fragment patterns. φEa116C contained an approximately 75-kb genome. φEa1, φEa7, φEa100, φEa125, and φEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40
E. amylovora
strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12
E. amylovora
strains isolated from raspberry fields (
Rubus
spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. φEa116C was more effective than the other phages at lysing
E. amylovora
strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10
5
CFU.
Bacteria
and
Archaea
Physically Associated with Gulf of Mexico Gas Hydrates
Authors: Lanoil Brian D. , Sassen Roger , La Duc Myron T. , Sweet Stephen T. , Nealson Kenneth H.
Published: July 2002

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The δ
13
C of hydrate-bound methane varied from −45.1‰ Peedee belemnite (PDB) to −74.7‰ PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 × 10
9
cells g
−1
) and gas hydrate (mean = 1.0 × 10
6
cells ml
−1
). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption.
Bacteria
were moderately diverse within AT425 and were dominated by gene sequences related to several groups of
Proteobacteria
, as well as
Actinobacteria
and low-G + C
Firmicutes
. In contrast, there was low diversity of
Archaea
, nearly all of which were related to methanogenic
Archaea
, with the majority specifically related to
Methanosaeta
spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.
Sorption of Fe (Hydr)Oxides to the Surface of
Shewanella putrefaciens
: Cell-Bound Fine-Grained Minerals Are Not Always Formed De Novo
Authors: Glasauer S. , Langley S. , Beveridge T. J.
Published: July 2002

Shewanella putrefaciens
, a gram-negative, facultative anaerobe, is active in the cycling of iron through its interaction with Fe (hydr)oxides in natural environments. Fine-grained Fe precipitates that are attached to the outer membranes of many gram-negative bacteria have most often been attributed to precipitation and growth of the mineral at the cell surface. Our study of the sorption of nonbiogenic Fe (hydr)oxides revealed, however, that large quantities of nanometer-scale ferrihydrite (hydrous ferric oxide), goethite (α-FeOOH), and hematite (α-Fe
2
O
3
) adhered to the cell surface. Attempts to separate suspensions of cells and minerals with an 80% glycerin cushion proved that the sorbed minerals were tightly attached to the bacteria. The interaction between minerals and cells resulted in the formation of mineral-cell aggregates, which increased biomass density and provided better sedimentation of mineral Fe compared to suspensions of minerals alone. Transmission electron microscopy observations of cells prepared by whole-mount, conventional embedding, and freeze-substitution methods confirmed the close association between cells and minerals and suggested that in some instances, the mineral crystals had even penetrated the outer membrane and peptidoglycan layers. Given the abundance of these mineral types in natural environments, the data suggest that not all naturally occurring cell surface-associated minerals are necessarily formed de novo on the cell wall.
Comparative Study of the Cyclization Reactions of Three Bacterial Cyclomaltodextrin Glucanotransferases
Authors: Terada Yoshinobu , Sanbe Haruyo , Takaha Takeshi , Kitahata Sumio , Koizumi Kyoko , Okada Shigetaka
Published: July 2002

The actions of cyclomaltodextrin glucanotransferases (CGTase; EC
2.4.1.19
) from alkalophilic
Bacillus
sp. strain A2-5a (A2-5a CGTase),
Bacillus macerans
(
Bmac
CGTase), and
Bacillus stearothermophilus
(
Bste
CGTase) on amylose were investigated. All three enzymes produced large cyclic α-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CD
n
, a cycloamylose with a degree of polymerization of
n
). A2-5a CGTase produced 10 times more CD7, while
Bmac
CGTase produced 34 times more CD6 than other cycloamyloses.
Bste
CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.
Growth Interactions during Bacterial Colonization of Seedling Rootlets
Authors: De Bellis P. , Ercolani G. L.
Published: July 2002

Rootlet elongation and bacterial growth on rootlets were determined after inoculation of cucumber and spinach seedlings with
Pseudomonas
strains differing in production of siderophores and HCN. Siderophore producers grew more profusely than nonproducers on both species and promoted rootlet elongation on cucumber. Coinoculation of siderophore producers and nonproducers resulted in restricted growth of the latter. The total populations of nonproducers of HCN in the presence of HCN producers were not decreased, but the tenacity of their association with the rootlet surface was altered.
Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination
Authors: Siciliano Steven D. , Fortin Nathalie , Mihoc Anca , Wisse Gesine , Labelle Suzanne , Beaumier Danielle , Ouellette Danielle , Roy Real , Whyte Lyle G. , Banks M. Kathy , Schwab Paul , Lee Ken , Greer Charles W.
Published: July 2002

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (
alkB
) and naphthalene dioxygenase (
ndoB
), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (
ntdAa
) and nitrotoluene monooxygenase (
ntnM
), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of
ndoB
-positive endophytes in
Scirpus pungens
, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of
alkB
- or
ndoB
-positive endophytes in
Festuca arundinacea
were correlated with the concentration of creosote in the soil but not with the numbers of
alkB
- or
ndoB
-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.
Cloning and Expression of the Benzoate Dioxygenase Genes from
Rhodococcus
sp. Strain 19070
Authors: Haddad Sandra , Eby D. Matthew , Neidle Ellen L.
Published: July 2002

The
bopXYZ
genes from the gram-positive bacterium
Rhodococcus
sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled
Escherichia coli
to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic
cis
-diol or catechol, respectively. This expression system also rapidly transformed
m
-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in
Rhodococcus
sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both
meta
- and
ortho
-cleavage degradative pathways, which are associated in pseudomonads with the
xyl
and
ben
genes, respectively. Open reading frames downstream of
bopXYZ
, designated
bopL
and
bopK
, resembled genes encoding
cis
-diol dehydrogenases and benzoate transporters, respectively. The
bop
genes were in the same order as the chromosomal
ben
genes of
P. putida
PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in
E. coli
yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome
c
reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.
Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling
Authors: Schmalenberger Achim , Schwieger Frank , Tebbe Christoph C.
Published: July 2002

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (
Zea mays
), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (α, β, and γ subgroups) and members of the genus
Paenibacillus
(low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking “universal” primers can affect a PCR-based microbial community analysis.
Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from
Mycobacterium
sp. Strain PYR-1
Authors: Khan Ashraf A. , Wang Rong-Fu , Cao Wei-Wen , Doerge Daniel R. , Wennerstrom David , Cerniglia Carl E.
Published: July 2002

Mycobacterium
sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of
Mycobacterium
sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of
Mycobacterium
sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from
Rhodococcus
spp. and
Nocardioides
spp. The genes from
Mycobacterium
sp. strain PYR-1 were subcloned and overexpressed in
Escherichia coli
with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.
Horizontal Transfer of a Multi-Drug Resistance Plasmid between Coliform Bacteria of Human and Bovine Origin in a Farm Environment
Authors: Oppegaard Hanne , Steinum Terje M. , Wasteson Yngvild
Published: July 2002

Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment.
Development of a Virus Concentration Method and Its Application to Detection of Enterovirus and Norwalk Virus from Coastal Seawater
Authors: Katayama Hiroyuki , Shimasaki Akihiro , Ohgaki Shinichiro
Published: July 2002

We developed a new procedure for concentration of enteric viruses from water using a negatively charged membrane. Rinsing the membrane with 0.5 mM H
2
SO
4
(pH 3.0) in order to elute cations prior to viral elution with 1 mM NaOH (pH 10.5) promoted poliovirus recovery yields from 33 to 95% when applied to pure water and 38 to 89% when applied to natural seawater from Tokyo Bay, Japan, respectively. This method showed average recovery yields of spiked poliovirus of 62% (
n
= 8) from 1 liter of artificial seawater. This method showed higher recovery yields (>61%) than that of the conventional method using positively charged membrane (6%) when applied to seawater. This method is also free from beef extract elution, which has an inhibitory effect in the subsequent viral genome detection by reverse transcription-PCR. Naturally occurring Norwalk viruses from 2 liters of Tokyo Bay water in winter and infectious enteroviruses from 2 liters of recreational coastal seawater in summer were detected by using this viral concentration method.
Characterization of a Chitinase Gene from
Stenotrophomonas maltophilia
Strain 34S1 and Its Involvement in Biological Control
Authors: Kobayashi Donald Y. , Reedy Ralph M. , Bick JulieAnn , Oudemans Peter V.
Published: July 2002

A chitinase gene was cloned on a 2.8-kb DNA fragment from
Stenotrophomonas maltophilia
strain 34S1 by heterologous expression in
Burkholderia cepacia
. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of
S. maltophilia
strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by
chiA
. Marker exchange mutagenesis of
chiA
resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of
S. maltophilia
.
Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters
Authors: Sinton Lester W. , Hall Carollyn H. , Lynch Philippa A. , Davies-Colley Robert J.
Published: July 2002

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci,
Escherichia coli
, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (
k
S
) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (
k
D
) rates in enclosed (control) chambers. The overall
k
S
ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥
E. coli
> somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.
Production of N2through Anaerobic Ammonium Oxidation Coupled to Nitrate Reduction in Marine Sediments
Authors: Thamdrup Bo , Dalsgaard Tage
Published: July 2002

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N2, thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N2production in marine sediments. Incubations with15N-labeled nitrate or ammonium demonstrated that during this process, N2is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N2production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N2production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N2production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N2, anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.
Effects of Amendment with Ferrihydrite and Gypsum on the Structure and Activity of Methanogenic Populations in Rice Field Soil
Authors: Lueders Tillmann , Friedrich Michael W.
Published: July 2002

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO
4
 · 2H
2
O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 μM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of
Methanosarcina
populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of
Methanosaeta
spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels (∼0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 μM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.
Proteomic Analysis Reveals Differential Protein Expression by
Bacillus cereus
during Biofilm Formation
Authors: Oosthuizen Marinda C. , Steyn Bridgitta , Theron Jacques , Cosette Pascal , Lindsay Denise , von Holy Alexander , Brözel Volker S.
Published: July 2002

Bacillus cereus
, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods.
B. cereus
DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and
l
-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both σ
B
and σ
H
, could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.
Elemental Composition (C, N, P) and Cell Volume of Exponentially Growing and Nutrient-Limited Bacterioplankton
Authors: Vrede Katarina , Heldal Mikal , Norland Svein , Bratbak Gunnar
Published: July 2002

Marine bacterioplankton were isolated and grown in batch cultures until their growth became limited by organic carbon (C), nitrogen (N), or phosphorus (P). Samples were taken from the cultures at both the exponential and stationary phases. The elemental composition of individual bacterial cells was analyzed by X-ray microanalysis with an electron microscope. The cell size was also measured. The elemental content was highest in exponentially growing cells (149 ± 8 fg of C cell
−1
, 35 ± 2 fg of N cell
−1
, and 12 ± 1 fg of P cell
−1
; average of all isolates ± standard error). The lowest C content was found in C-limited cells (39 ± 3 fg of C cell
−1
), the lowest N content in C- and P-limited cells (12 ± 1 and 12 ± 2 fg of N cell
−1
, respectively), and the lowest P content in P-limited cells (2.3 ± 0.6 fg of P cell
−1
). The atomic C:N ratios varied among treatments between 3.8 ± 0.1 and 9.5 ± 1.0 (average ± standard error), the C:P ratios between 35 ± 2 and 178 ± 28, and the N:P ratios between 6.7 ± 0.3 and 18 ± 3. The carbon-volume ratios showed large variation among isolates due to different types of nutrient limitation (from 51± 4 to 241 ± 38 fg of C μm
−1
; average of individual isolates and treatments ± standard error). The results show that different growth conditions and differences in the bacterial community may explain some of the variability of previously reported elemental and carbon-volume ratios.