bluuebunny/crossref_metadata_2025 huggingface dataset:
Electricity Production byGeobacter sulfurreducensAttached to Electrodes
Authors: Bond Daniel R. , Lovley Derek R.
Published: March 2003

Previous studies have suggested that members of theGeobacteraceaecan use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production,Geobacter sulfurreducenswas inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum ofG. sulfurreducenswas introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (Eo′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m2of electrode surface) or poised-potential (163 to 1,143 mA/m2) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such asG. sulfurreducensthat can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.
Evaluation of Potential Indicators of Viral Contamination in Shellfish and Their Applicability to Diverse Geographical Areas
Authors: Formiga-Cruz M. , Allard A. K. , Conden-Hansson A.-C. , Henshilwood K. , Hernroth B. E. , Jofre J. , Lees D. N. , Lucena F. , Papapetropoulou M. , Rangdale R. E. , Tsibouxi A. , Vantarakis A. , Girones R.
Published: March 2003

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of
Escherichia coli
, F-specific RNA bacteriophages, and phages infecting
Bacteroides fragilis
strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where
E. coli
would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of
E. coli
or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.
Prokaryotic Homologs of the Eukaryotic 3-Hydroxyanthranilate 3,4-Dioxygenase and 2-Amino-3-Carboxymuconate-6-Semialdehyde Decarboxylase in the 2-Nitrobenzoate Degradation Pathway ofPseudomonas fluorescensStrain KU-7
Authors: Muraki Takamichi , Taki Masami , Hasegawa Yoshie , Iwaki Hiroaki , Lau Peter C. K.
Published: March 2003

The 2-nitrobenzoic acid degradation pathway ofPseudomonas fluorescensstrain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilatemeta-cleavage pathway genes. The gene cluster, designatednbaEXHJIGFCDR,is organized tightly and in the same direction. ThenbaCandnbaDgene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed inEscherichia coliand characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. InE. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe2+but is inhibited by other metal ions, such as Zn2+, Cu2+, and Cd2+. The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH inPseudomonassp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.
Bacterial rRNA Genes Associated with Soil Suppressiveness against the Plant-Parasitic Nematode
Heterodera schachtii
Authors: Yin Bei , Valinsky Lea , Gao Xuebiao , Becker J. Ole , Borneman James
Published: March 2003

The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode
Heterodera schachtii
. Since
H. schachtii
cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with
H. schachtii
cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with
H. schachtii
cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (
Actinobacteria
,
Cytophaga-Flexibacter-Bacteroides
, α-
Proteobacteria
, β-
Proteobacteria
, and γ-
Proteobacteria
) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the
H. schachtii
suppressiveness. When these three groups were examined with specific PCR analyses performed on
H. schachtii
cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several
Rhizobium
species and uncultured α-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this
Rhizobium
-like rDNA group with the
H. schachtii
suppressiveness.
Conidial Hydrophobins ofAspergillus fumigatus
Authors: Paris Sophie , Debeaupuis Jean-Paul , Crameri Reto , Carey Marilyn , Charlès Franck , Prévost Marie Christine , Schmitt Christine , Philippe Bruno , Latgé Jean Paul
Published: March 2003

The surface ofAspergillus fumigatusconidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. TheRODAgene was previously characterized, and ΔrodAconidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). TheRODBgene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp ofA. nidulans. ΔrodBconidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of ΔrodAconidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the ΔrodAΔrodBdouble mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.
Identification and Characterization of Two Novel Clostridial Bacteriocins, Circularin A and Closticin 574
Authors: Kemperman Robèr , Kuipers Anneke , Karsens Harma , Nauta Arjen , Kuipers Oscar , Kok Jan
Published: March 2003

Two novel antibacterial peptides of clostridial species were purified, N-terminally sequenced, and characterized. Moreover, their structural genes were identified. Closticin 574 is an 82-amino-acid bacteriocin produced byClostridium tyrobutyricumADRIAT 932. The supernatant of the producing strain showed a high level of activity against the indicator strainC. tyrobutyricum. The protein is synthesized as a preproprotein that is possibly secreted via the general secretion pathway, after which it is hydrolyzed at an Asp-Pro site. Circularin A is produced byClostridium beijerinckiiATCC 25752 as a prepeptide of 72 amino acids. Cleavage of the prepeptide between the third leucine and fourth valine residues followed by a head-to-tail ligation between the N and C termini creates a circular antimicrobial peptide of 69 amino acids. The unusually small circularin A leader peptide of three amino acids is cleaved off in this process. The supernatant ofC. beijerinckiiATCC 25752 showed a broad antibacterial activity range.
Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the β-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the
Acinetobacter
sp. Strain ADP1
pobA
Gene
Authors: Brzostowicz Patricia C. , Reams Andrew B. , Clark Todd J. , Neidle Ellen L.
Published: March 2003

Transcriptional control of carbon source preferences by
Acinetobacter
sp. strain ADP1 was assessed with a
pobA
::
lacZ
fusion during growth on alternative substrates. The
pobA
-encoded enzyme catalyzes the first step in the degradation of 4-hydroxybenzoate, a compound consumed rapidly as a sole carbon source. If additional aromatic carbon sources are available, 4-hydroxybenzoate consumption is inhibited by unknown mechanisms. As reported here, during growth on aromatic substrates,
pobA
was not expressed despite the presence of 4-hydroxybenzoate, an inducer that normally causes the PobR regulator to activate
pobA
transcription. Growth on organic acids such as succinate, fumarate, and acetate allowed higher levels of
pobA
expression. In each case,
pobA
expression increased at the end of the exponential growth phase. Complex transcriptional regulation controlled 4-hydroxybenzoate catabolism in multisubstrate environments. Additional studies focused on the wild-type preference for benzoate consumption prior to 4-hydroxybenzoate consumption. These compounds are degraded via the catechol and protocatechuate branches of the β-ketoadipate pathway, respectively. Here, mutants were characterized that degraded benzoate and 4-hydroxybenzoate concurrently. These mutants lacked the BenM and CatM transcriptional regulators that normally activate genes for benzoate catabolism. A model is presented in which BenM and CatM prevent
pobA
expression indirectly during growth on benzoate. These regulators may affect
pobA
expression by lowering the PcaK-mediated uptake of 4-hydroxybenzoate. Consistent with this model, BenM and CatM bound in vitro to an operator-promoter fragment controlling the expression of several
pca
genes, including
pcaK
. These studies provide the first direct evidence of transcriptional cross-regulation between the distinct but analogous branches of the β-ketoadipate pathway.
Tri1Encodes the Cytochrome P450 Monooxygenase for C-8 Hydroxylation during Trichothecene Biosynthesis inFusarium sporotrichioidesand Resides Upstream of Another NewTriGene
Authors: Meek Isaac B. , Peplow Andrew W. , Ake, Charles , Phillips T. D. , Beremand Marian N.
Published: March 2003

ManyFusariumspecies produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure. Previous studies with UV-inducedFusarium sporotrichioidesNRRL 3299 trichothecene mutants defined theTri1gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis. We have cloned and characterized theTri1gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase. The disruption ofTri1blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in thetri1UV-induced mutants MB1716 and MB1370. TheTri1disruptants and thetri1UV-induced mutants do not complement one another when coinoculated, and theTri1gene sequence restores T-2 toxin production in both MB1716 and MB1370. The DNA sequence flankingTri1contains another newTrigene. Thus,Tri1encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster.
Respiration of
13
C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of
13
C-Labeled Soil DNA
Authors: Padmanabhan P. , Padmanabhan S. , DeRito C. , Gray A. , Gannon D. , Snape J. R. , Tsai C. S. , Park W. , Jeon C. , Madsen E. L.
Published: March 2003

Our goal was to develop a field soil biodegradation assay using
13
C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived
13
C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of
13
C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for
13
CO
2
respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of
13
CO
2
emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF
6
, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired
13
CO
2
. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of
13
CO
2
released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of
13
C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with
13
C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of
Arthrobacter
,
Pseudomonas
,
Acinetobacter
,
Massilia
,
Flavobacterium
, and
Pedobacter
spp. for glucose;
Pseudomonas
,
Pantoea
,
Acinetobacter
,
Enterobacter
,
Stenotrophomonas
, and
Alcaligenes
spp. for phenol;
Pseudomonas
,
Acinetobacter
, and
Variovorax
spp. for naphthalene; and
Acinetobacter
,
Enterobacter
,
Stenotrophomonas
, and
Pantoea
spp. for caffeine.
Improved Anaerobic Use of Arginine by
Saccharomyces cerevisiae
Authors: Martin Olga , Brandriss Marjorie C. , Schneider Gisbert , Bakalinsky Alan T.
Published: March 2003

Anaerobic arginine catabolism in
Saccharomyces cerevisiae
was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A
pro3 ure2
strain expressing a
PGK1
promoter-driven
PUT2
allele encoding Δ
1
-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.
Infectivity of RNA from Inactivated Poliovirus
Authors: Nuanualsuwan Suphachai , Cliver Dean O.
Published: March 2003

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.
The Genes Coding for Enterocin EJ97 Production by
Enterococcus faecalis
EJ97 Are Located on a Conjugative Plasmid
Authors: Sánchez-Hidalgo Marina , Maqueda Mercedes , Gálvez Antonio , Abriouel Hikmate , Valdivia Eva , Martínez-Bueno Manuel
Published: March 2003

Enterococcus faecalis
EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain
E. faecalis
EJ97 to
E. faecalis
OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb
Eco
RI-
Bgl
II DNA fragment of pEJ97. This region was cloned and sequenced. It contains the
ej97A
structural gene plus three open reading frames (ORFs) (
ej97B
,
ej97C
, and
ej97D
) and three putative ORFs transcribed in the opposite direction (
orfA
,
orfB
, and
orfC
). The gene
ej97A
translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of
ej97B
(Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of
ej97C
(Ej97C) and
ej97D
(Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the
ej97
module, which have been identified as a transposon-like structure (
tnp
). ORF1 shows similarities to transposase of the
Lactococcus lactis
element IS
S1
and is up to 50% identical to IS
1216
. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of IS
S1
and IS
1216
. ORF6 (
resEJ97
) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from
Staphylococcus aureus
and
E. faecalis.
These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of
E. faecalis
, suggesting that they have a common origin.
Enterotoxigenicity and Genetic Relatedness ofClostridium perfringensIsolates from Retail Foods in the United States
Authors: Lin Yuan-Tong , Labbe Ronald
Published: March 2003

Clostridium perfringensis a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45°C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of theC. perfringensenterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessedcpa. None of the isolates were identified as carrying thecpegene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity ofcpe+strains in retail foods and the genetic diversity among nonoutbreak strains.
Characterization of the
Streptomyces
sp. Strain C5
snp
Locus and Development of
snp
-Derived Expression Vectors
Authors: DeSanti Charles L. , Strohl William R.
Published: March 2003

The
Streptomyces
sp. strain C5
snp
locus is comprised of two divergently oriented genes:
snpA
, a metalloproteinase gene, and
snpR
, which encodes a LysR-like activator of
snpA
transcription. The transcriptional start point of
snpR
is immediately downstream of a strong T-N
11
-A inverted repeat motif likely to be the SnpR binding site, while the
snpA
transcriptional start site overlaps the ATG start codon, generating a leaderless
snpA
transcript. By using the
aphII
reporter gene of pIJ486 as a reporter, the plasmid-borne
snpR
-activated
snpA
promoter was ca. 60-fold more active than either the nonactivated
snpA
promoter or the
melC1
promoter of pIJ702. The
snpR
-activated
snpA
promoter produced reporter protein levels comparable to those of the up-mutated
ermE∗
promoter. The SnpR-activated
snpA
promoter was built into a set of transcriptional and translational fusion expression vectors which have been used for the intracellular expression of numerous daunomycin biosynthesis pathway genes from
Streptomyces
sp. strain C5 as well as the expression and secretion of soluble recombinant human endostatin.
N
2
O-Producing Microorganisms in the Gut of the Earthworm
Aporrectodea caliginosa
Are Indicative of Ingested Soil Bacteria
Authors: Ihssen Julian , Horn Marcus A. , Matthies Carola , Gößner Anita , Schramm Andreas , Drake Harold L.
Published: March 2003

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N
2
O) and (ii) to characterize the N
2
O-producing bacteria of the earthworm gut. The production of N
2
O in the gut of garden soil earthworms (
Aporrectodea caliginosa
) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N
2
O were transient products when supplemental nitrate was reduced to N
2
by gut content homogenates. In contrast, nitrite and N
2
O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N
2
O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N
2
O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class
Proteobacteria
or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N
2
O or N
2
. The initial N
2
O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N
2
O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N
2
O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.
The Earthworm Gut: an Ideal Habitat for Ingested N2O-Producing Microorganisms
Authors: Horn Marcus A. , Schramm Andreas , Drake Harold L.
Published: March 2003

The in vivo production of nitrous oxide (N2O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N2O-producing soil bacteria. In situ measurement of N2O and O2with microsensors demonstrated that the earthworm gut is anoxic and the site of N2O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N2O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N2O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N2O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N2O, and addition of buffer or ammonium had essentially no effect on the initial N2O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N2O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N2O-producing soil bacteria during gut passage.
Synthesis of Imidazol-2-yl Amino Acids by Using Cells from Alkane-Oxidizing Bacteria
Authors: Mikolasch Annett , Hammer Elke , Schauer Frieder
Published: March 2003

Sixty-one strains of alkane-oxidizing bacteria were tested for their ability to oxidize
N
-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide to imidazol-2-yl amino acids applicable for pharmaceutical purposes. After growth with
n
-alkane, 15 strains formed different imidazol-2-yl amino acids identified by chemical structure analysis (mass and nuclear magnetic resonance spectrometry). High yields of imidazol-2-yl amino acids were produced by the strains
Gordonia rubropertincta
SBUG 105,
Gordonia terrae
SBUG 253,
Nocardia asteroides
SBUG 175,
Rhodococcus erythropolis
SBUG 251, and
Rhodococcus erythropolis
SBUG 254. Biotransformation occurred via oxidation of the alkyl side chain and produced 1-acetylamino-4-phenylimidazol-2-yl-6-aminohexanoic acid and the butanoic acid derivative. In addition, the acetylamino group of these products and of the substrate was transformed to an amino group. The product pattern as well as the transformation pathway of
N
-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide differed in the various strains used.
Biogeochemical Evidence that Thermophilic Archaea Mediate the Anaerobic Oxidation of Methane
Authors: Schouten Stefan , Wakeham Stuart G. , Hopmans Ellen C. , Sinninghe Damsté Jaap S.
Published: March 2003

Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically. The presence of strongly
13
C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30°C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites. The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.
Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease
Authors: Brinig Mary M. , Lepp Paul W. , Ouverney Cleber C. , Armitage Gary C. , Relman David A.
Published: March 2003

Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site. Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria. TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR. Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization. The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% ± 0.1%) than in either healthy sites (0.21% ± 0.05%,
P
< 0.01) or sites with severe periodontitis (0.29% ± 0.06%,
P
< 0.05). One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples. These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis.
Early Events in the
Fusarium verticillioides
-Maize Interaction Characterized by Using a Green Fluorescent Protein-Expressing Transgenic Isolate
Authors: Oren Liat , Ezrati Smadar , Cohen David , Sharon Amir
Published: March 2003

The infection of maize by
Fusarium verticillioides
can result in highly variable disease symptoms ranging from asymptomatic plants to severe rotting and wilting. We produced
F. verticillioides
green fluorescent protein-expressing transgenic isolates and used them to characterize early events in the
F. verticillioides-
maize interaction that may affect later symptom appearance. Plants grown in
F. verticillioides-
infested soil were smaller and chlorotic. The fungus colonized all of the underground parts of a plant but was found primarily in lateral roots and mesocotyl tissue. In some mesocotyl cells, conidia were produced within 14 to 21 days after infection. Intercellular mycelium was detected, but additional cells were not infected until 21 days after planting. At 25 to 30 days after planting, the mesocotyl and main roots were heavily infected, and rotting developed in these tissues. Other tissues, including the adventitious roots and the stem, appeared to be healthy and contained only a small number of hyphae. These results imply that asymptomatic systemic infection is characterized by a mode of fungal development that includes infection of certain tissues, intercellular growth of a limited number of fungal hyphae, and reproduction of the fungus in a few cells without invasion of other cells. Development of visibly rotted tissue is associated with massive production of fungal mycelium and much less organized growth.
Mass Transport of Macromolecules within an In Vitro Model of Supragingival Plaque
Authors: Thurnheer Thomas , Gmür Rudolf , Shapiro Stuart , Guggenheim Bernhard
Published: March 2003

The aim of this study was to examine the diffusion of macromolecules through an in vitro biofilm model of supragingival plaque. Polyspecies biofilms containing
Actinomyces naeslundii
,
Fusobacterium nucleatum
,
Streptococcus oralis
,
Streptococcus sobrinus
,
Veillonella dispar
, and
Candida albicans
were formed on sintered hydroxyapatite disks and then incubated at room temperature for defined periods with fluorescent markers with molecular weights ranging from 3,000 to 900,000. Subsequent examination by confocal laser scanning microscopy revealed that the mean square penetration depths for all tested macromolecules except immunoglobulin M increased linearly with time, diffusion coefficients being linearly proportional to the cube roots of the molecular weights of the probes (range, 10,000 to 240,000). Compared to diffusion in bulk water, diffusion in the biofilms was markedly slower. The rate of diffusion for each probe appeared to be constant and not a function of biofilm depth. Analysis of diffusion phenomena through the biofilms suggested tortuosity as the most probable explanation for retarded diffusion. Selective binding of probes to receptors present in the biofilms could not explain the observed extent of retardation of diffusion. These results are relevant to oral health, as selective attenuated diffusion of fermentable carbohydrates and acids produced within dental plaque is thought to be essential for the development of carious lesions.
Role of
Rhodobacter
sp. Strain PS9, a Purple Non-Sulfur Photosynthetic Bacterium Isolated from an Anaerobic Swine Waste Lagoon, in Odor Remediation
Authors: Do Young S. , Schmidt Thomas M. , Zahn James A. , Boyd Eric S. , de la Mora Arlene , DiSpirito Alan A.
Published: March 2003

Temporal pigmentation changes resulting from the development of a purple color in anaerobic swine waste lagoons were investigated during a 4-year period. The major purple photosynthetic bacterium responsible for these color changes and the corresponding reductions in odor was isolated from nine photosynthetic lagoons. By using morphological, physiological, and phylogenetic characterization methods we identified the predominant photosynthetic bacterium as a new strain of
Rhodobacter
, designated
Rhodobacter
sp. strain PS9.
Rhodobacter
sp. strain PS9 is capable of photoorganotrophic growth on a variety of organic compounds, including all of the characteristic volatile organic compounds (VOC) responsible for the odor associated with swine production facilities (J. A. Zahn, A. A. DiSpirito, Y. S. Do, B. E. Brooks, E. E. Copper, and J. L. Hatfield, J. Environ. Qual.
30:
624-634, 2001). The seasonal variations in airborne VOC emitted from waste lagoons showed that there was a 80 to 93% decrease in the concentration of VOC during a photosynthetic bloom. During the height of a bloom, the
Rhodobacter
sp. strain PS9 population accounted for 10% of the total community and up to 27% of the eubacterial community based on 16S ribosomal DNA signals. Additional observations based on seasonal variations in meteorological, biological, and chemical parameters suggested that the photosynthetic blooms of
Rhodobacter
sp. strain PS9 were correlated with lagoon water temperature and with the concentrations of sulfate and phosphate. In addition, the photosynthetic blooms of
Rhodobacter
sp. strain PS9 were inversely correlated with the concentrations of protein and fluoride.
Natural Genetic Transformation in Monoculture
Acinetobacter
sp. Strain BD413 Biofilms
Authors: Hendrickx Larissa , Hausner Martina , Wuertz Stefan
Published: March 2003

Horizontal gene transfer by natural genetic transformation in
Acinetobacter
sp. strain BD413 was investigated by using
gfp
carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of
Acinetobacter
sp. strain BD413.
Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of
Vibrio cholerae
Authors: Li Manrong , Kotetishvili Mamuka , Chen Yuansha , Sozhamannan Shanmuga
Published: March 2003

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of
Vibrio cholerae
: cholera toxin encoded by the
ctxAB
genes and toxin-coregulated pilus encoded by the
tcpA
gene. The
ctx
genes reside in the genome of a filamentous phage (CTXφ), and the
tcpA
gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIφ. In order to determine the prevalence of horizontal transfer of VPI and CTXφ among nonepidemic (non-O1 and non-O139 serogroups)
V. cholerae
, 300 strains of both clinical and environmental origin were screened for the presence of
tcpA
and
ctxAB
. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI
+
strains have also acquired the CTXφ. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXφ prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXφ prophage types characterized by novel repressor (
rstR
) and
ctxAB
genes and VPIs with novel
tcpA
genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXφ independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.
Quorum-Sensing System and Stationary-Phase Sigma Factor (
rpoS
) of the Onion Pathogen
Burkholderia cepacia
Genomovar I Type Strain, ATCC 25416
Authors: Aguilar Claudio , Bertani Iris , Venturi Vittorio
Published: March 2003

Bacterial strains belonging to
Burkholderia cepacia
can be human opportunistic pathogens, plant pathogens, and plant growth promoting and have remarkable catabolic activity.
B. cepacia
consists of several genomovars comprising what is now known as the
B. cepacia
complex. Here we report the quorum-sensing system of a genomovar I onion rot type strain ATCC 25416. Quorum sensing is a cell-density-dependent regulatory response which involves the production of
N
-acyl homoserine lactone (HSL) signal molecules. The
cep
locus has been inactivated in the chromosome, and it has been shown that CepI is responsible for the biosynthesis of an
N
-hexanoyl HSL (C
6
-HSL) and an
N
-octanoyl HSL (C
8
-HSL) and that the
cep
locus regulates protease production as well as onion pathogenicity via the expression of a secreted polygalacturonase. A
cep
-
lacZ
-based sensor plasmid has been constructed and used to demonstrate that CepR responded to C
6
-HSL with only 15% of the molar efficiency of C
8
-HSL, that a
cepR
knockout mutant synthesized 70% less HSLs, and that CepR responded best towards long-chain HSLs. In addition, we also report the cloning and characterization of the stationary-phase sigma factor gene
rpoS
of
B. cepacia
ATCC 25416. It was established that quorum sensing in
B. cepacia
has a negative effect on
rpoS
expression as determined by using an
rpoS
-
lacZ
transcriptional fusion; on the other hand,
rpoS
-null mutants displayed no difference in the accumulation of HSL signal molecules.
In Situ Accessibility of Small-Subunit rRNA of Members of the Domains
Bacteria
,
Archaea
, and
Eucarya
to Cy3-Labeled Oligonucleotide Probes
Authors: Behrens Sebastian , Rühland Caroline , Inácio João , Huber Harald , Fonseca Á. , Spencer-Martins I. , Fuchs Bernhard M. , Amann Rudolf
Published: March 2003

Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of
Escherichia coli
(gamma
Proteobacteria
,
Bacteria
) with the phylogenetically distantly related organisms
Pirellula
sp. strain 1 (
Planctomycetes
,
Bacteria
) and
Metallosphaera sedula
(
Crenarchaeota
,
Archaea
) and the 18S rRNA accessibility of
Saccharomyces cerevisiae
(
Eucarya
). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For
Pirellula
sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on
M. sedula
were grouped into class V and VI (46% of the whole data set). For
E. coli
, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of
S. cerevisiae
.
DNA Microarray Analyses of the Long-Term Adaptive Response ofEscherichia colito Acetate and Propionate
Authors: Polen T. , Rittmann D. , Wendisch V. F. , Sahm H.
Published: March 2003

In its natural environment,Escherichia coliis exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations. DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH. The adaptive responses to acetate and propionate were similar and involved genes in three categories. First, the RNA levels for chemotaxis and flagellum genes increased. Accordingly, the expression of chromosomalfliC′-′lacZandflhDC′-′lacZfusions and swimming motility increased after adaptation to acetate or propionate. Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate. Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response. Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes. The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response ofE. colito either compound.