bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomarker Evidence for Widespread Anaerobic Methane Oxidation in Mediterranean Sediments by a Consortium of Methanogenic Archaea and Bacteria
Authors: Pancost Richard D. , Sinninghe Damsté Jaap S. , de Lint Saskia , van der Maarel Marc J. E. C. , Gottschal Jan C. ,   The Medinaut Shipboard Scientific Party
Published: July 2002

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible
Nautile
. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in
13
C (δ
13
C values are as low as −95‰). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of
13
C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.
rRNA Operon Copy Number Reflects Ecological Strategies of Bacteria
Authors: Klappenbach Joel A. , Dunbar John M. , Schmidt Thomas M.
Published: July 2002

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes (

x

¯

= 5.4) became dominant, whereas populations with fewer rRNA genes (

x

¯

= 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.
Effects of Acid Adaptation, Product pH, and Heating on Survival of
Escherichia coli
O157:H7 in Pepperoni
Authors: R. Riordan Denise C. , Duffy Geraldine , Sheridan James J. , Whiting Richard C. , Blair Ian S. , McDowell David A.
Published: July 2002

The thermotolerance of
E. coli
O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of
E. coli
O157:H7 in pepperoni was applied in the development of heating processes designed to reduce
E. coli
O157:H7 numbers therein by 5 log
10
units.
Genetic and Biochemical Characterization of a Highly Thermostable α-
l
-Arabinofuranosidase from
Thermobacillus xylanilyticus
Authors: Debeche Takoua , Cummings Nicola , Connerton Ian , Debeire Philippe , O'Donohue Michael J.
Published: July 2002

The gene encoding an α-
l
-arabinofuranosidase from
Thermobacillus xylanilyticus
D3, AbfD3, was isolated. Characterization of the purified recombinant α-
l
-arabinofuranosidase produced in
Escherichia coli
revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.
Characterization of a Novel Integrative Element, ICE
St1
, in the Lactic Acid Bacterium
Streptococcus thermophilus
Authors: Burrus Vincent , Roussel Yvonne , Decaris Bernard , Guédon Gérard
Published: July 2002

The 35.5-kb ICE
St1
element of
Streptococcus thermophilus
CNRZ368 is bordered by a 27-bp repeat and integrated into the 3′ end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn
5276
and Tn
5252
. The integrase was found to be involved in a site-specific excision of a circular form. ICE
St1
also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn
916
. Therefore, ICE
St1
could be or could be derived from an integrative conjugative element.
Coexistence of Two Different O Demethylation Systems in Lignin Metabolism by
Sphingomonas paucimobilis
SYK-6: Cloning and Sequencing of the Lignin Biphenyl-Specific
O
-Demethylase (LigX) Gene
Authors: Sonoki Tomonori , Obi Takahiro , Kubota Sachiko , Higashi Motoo , Masai Eiji , Katayama Yoshihiro
Published: July 2002

Sphingomonas paucimobilis
SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5,5′-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designated
ligX
) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in
S. paucimobilis
SYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6.
S. paucimobilis
SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.
Biotransformation of Hydroxylaminobenzene and Aminophenol by
Pseudomonas putida
2NP8 Cells Grown in the Presence of 3-Nitrophenol
Authors: Zhao Jian-Shen , Singh Ajay , Huang Xiao-Dong , Ward Owen P.
Published: July 2002

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of
Pseudomonas putida
2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone,
N
-acetyl-4-aminophenol,
N
-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia,
N
-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of
P. putida
2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in
P. putida
2NP8, in which all of the possible intermediates are postulated.
Differential Effects of Permeating and Nonpermeating Solutes on the Fatty Acid Composition of
Pseudomonas putida
Authors: Halverson Larry J. , Firestone Mary K.
Published: July 2002

We examined the effect of reduced water availability on the fatty acid composition of
Pseudomonas putida
strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that −1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas −1.0-MPa NaCl-treated or control cells did not. At the range of water potential (−0.25 to −1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of
trans
and
cis
isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the −0.25-MPa water potential imposed by NaCl or PEG 200 had a higher
trans
:
cis
ratio than −0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of
trans
isomers, resulting in a higher
trans
:
cis
ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in −1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of
trans
fatty acids with a corresponding increase in the
cis
isomer. The decrease in the
trans/cis
ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.
Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments
Authors: King Jeffrey K. , Kostka Joel E. , Frischer Marc E. , Saunders F. Michael
Published: July 2002

Differences in methylmercury (CH
3
Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (
Desulfovibrio desulfuricans
,
Desulfobulbus propionicus
,
Desulfococcus multivorans
,
Desulfobacter
sp. strain BG-8, and
Desulfobacterium
sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family
Desulfobacteriaceae
) than in pure cultures of members of groups that are not able to use acetate (e.g., the family
Desulfovibrionaceae
). Little or no Hg methylation was observed in cultures of
Desulfobacterium
or
Desulfovibrio
strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the
Desulfobacterium
and
Desulfobacter
groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family
Desulfobacteriaceae
have a greater potential to methylate Hg than members of the family
Desulfovibrionaceae
have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH
3
Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH
3
Hg formation and for developing remediation strategies for Hg-contaminated sediments.
Cellulose Catabolism by
Clostridium cellulolyticum
Growing in Batch Culture on Defined Medium
Authors: Desvaux Mickaël , Guedon Emmanuel , Petitdemange Henri
Published: July 2002

A reinvestigation of cellulose degradation by
Clostridium cellulolyticum
in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter
−1
, acetate, ethanol, H
2
, and CO
2
were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H
2
and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter
−1
, the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831–1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of
C. cellulolyticum
. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter
−1
), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter
−1
), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter
−1
and 45% cellulose degradation within 18 days.
Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats
Authors: Skirnisdottir Sigurlaug , Hreggvidsson Gudmundur O. , Hjörleifsdottir Sigridur , Marteinsson Viggo T. , Petursdottir Solveig K. , Holst Olle , Kristjansson Jakob K.
Published: July 2002

In solfataric fields in southwestern Iceland, neutral and sulfide-rich hot springs are characterized by thick bacterial mats at 60 to 80°C that are white or yellow from precipitated sulfur (sulfur mats). In low-sulfide hot springs in the same area, grey or pink streamers are formed at 80 to 90°C, and a
Chloroflexus
mat is formed at 65 to 70°C. We have studied the microbial diversity of one sulfur mat (high-sulfide) hot spring and one
Chloroflexus
mat (low-sulfide) hot spring by cloning and sequencing of small-subunit rRNA genes obtained by PCR amplification from mat DNA. Using 98% sequence identity as a cutoff value, a total of 14 bacterial operational taxonomic units (OTUs) and 5 archaeal OTUs were detected in the sulfur mat; 18 bacterial OTUs were detected in the
Chloroflexus
mat. Although representatives of novel divisions were found, the majority of the sequences were >95% related to currently known sequences. The molecular diversity analysis showed that
Chloroflexus
was the dominant mat organism in the low-sulfide spring (1 mg liter
−1
) below 70°C, whereas
Aquificales
were dominant in the high-sulfide spring (12 mg liter
−1
) at the same temperature. Comparison of the present data to published data indicated that there is a relationship between mat type and composition of
Aquificales
on the one hand and temperature and sulfide concentration on the other hand.
Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading
Comamonas testosteroni
Strain, I2
gfp
Authors: Boon Nico , Goris Johan , De Vos Paul , Verstraete Willy , Top Eva M.
Published: July 2002

A strain identified as
Comamonas testosteroni
I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal
meta
-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the
gfp
gene and designated I2
gfp
. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.
Engineering of the Pyruvate Dehydrogenase Bypass inSaccharomyces cerevisiae: Role of the Cytosolic Mg2+and Mitochondrial K+Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation
Authors: Remize Fabienne , Andrieu Emilie , Dequin Sylvie
Published: July 2002

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate bySaccharomyces cerevisiaeduring alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with thePDC1gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressingACS2and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies ofALD6, encoding the cytosolic Mg2+-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion ofALD4, encoding the mitochondrial K+-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, theald6 ald4double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of theALDfamily.
Light and the Transcriptional Response of the Microcystin Biosynthesis Gene Cluster
Authors: Kaebernick Melanie , Neilan Brett A. , Börner Thomas , Dittmann Elke
Published: July 2002

Microcystin, a hepatotoxin known to be the cause of animal and human deaths, is produced by the bloom-forming cyanobacteriumMicrocystis aeruginosain freshwater bodies worldwide. The toxin is produced nonribosomally via a multifunctional enzyme complex, consisting of both peptide synthetase and polyketide synthase modules coded for by themcygene cluster. The recent identification of themcygenes in the production of microcystin synthetase for the first time provides an avenue to study the regulation of microcystin production at a genetic level. In this study,M. aeruginosaPCC7806 was grown either under continuous light of various intensities or under low light with subsequent short-term exposure to different light intensities and qualities and various stress factors. RNase protection assays were employed to observe the level ofmcyBandmcyDtranscription under each condition. BothmcyBandmcyDtranscript levels were increased under high light intensities and red light. Blue light and certain artificial stress factors (methylviologen and NaCl) led to reduced transcript amounts. There appeared to be two light thresholds, between dark and low light (16 μmol of photons m−2s−1), and medium (31 μmol of photons m−2s−1) and high light (68 μmol of photons m−2s−1), at which a significant increase in transcription occurred. Our findings show that the effect of light on microcystin synthetase production is due to light quality and is initiated at certain threshold intensities, which are not necessarily reflected by observed intracellular toxin bioactivity.
Effect of Field Inoculation with
Sinorhizobium meliloti
L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (
Medicago sativa
) and a Non-Target Plant (
Chenopodium album
)—Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria
Authors: Schwieger Frank , Tebbe Christoph C.
Published: July 2002

Fourteen weeks after field release of luciferase gene-tagged
Sinorhizobium meliloti
L33 in field plots seeded with
Medicago sativa
, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of
M. sativa
plants,
S. meliloti
L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of
M. sativa
and
Chenopodium album
, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the
Proteobacteria
(α, β, and γ subgroups), members of the
Cytophaga-Flavobacterium
group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of
M. sativa
, by inoculation. The most abundant isolates were related to
Variovorax
sp.,
Arthrobacter ramosus
, and
Acinetobacter calcoaceticus
. In the rhizosphere of
M. sativa
, inoculation reduced the numbers of cells of
A. calcoaceticus
and members of the genus
Pseudomonas
and increased the number of rhizobia. Cultivation-independent PCR–single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except
Variovorax
species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.
Purification and Characterization of an Extracellular Poly(
l
-Lactic Acid) Depolymerase from a Soil Isolate,
Amycolatopsis
sp. Strain K104-1
Authors: Nakamura Kohei , Tomita Toshio , Abe Naoki , Kamio Yoshiyuki
Published: July 2002

Poly(
l
-lactic acid) (PLA)-degrading
Amycolatopsis
sp. strains K104-1 and K104-2 were isolated by screening 300 soil samples for the ability to form clear zones on the PLA-emulsified mineral agar plates. Both of the strains assimilated >90% of emulsified 0.1% (wt/vol) PLA within 8 days under aerobic conditions. A novel PLA depolymerase with a molecular weight of 24,000 was purified to homogeneity from the culture supernatant of strain K104-1. The purified enzyme degraded high-molecular-weight PLA in emulsion and in solid film, ultimately forming lactic acid. The optimum pH for the enzyme activity was 9.5, and the optimum temperature was 55 to 60°C. The PLA depolymerase also degraded casein and fibrin but did not hydrolyze collagen type I, triolein, tributyrin, poly(β-hydroxybutyrate), or poly(ε-caprolactone). The PLA-degrading and caseinolytic activities of the enzyme were inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but were not significantly affected by soybean trypsin inhibitor,
N
-tosyl-
l
-lysyl chloromethyl ketone,
N
-tosyl-
l
-phenylalanyl chloromethyl ketone, and
Streptomyces
subtilisin inhibitor. Thus,
Amycolatopsis
sp. strain K104-1 excretes the unique PLA-degrading and fibrinolytic serine enzyme, utilizing extracellular polylactide as a sole carbon source.
Surface Interactions between
Escherichia coli
and Hemocytes of the Mediterranean Mussel
Mytilus galloprovincialis
Lam. Leading to Efficient Bacterial Clearance
Authors: Canesi Laura , Pruzzo Carla , Tarsi Renato , Gallo Gabriella
Published: July 2002

The role of type 1 fimbriae in the interactions between
Escherichia coli
and
Mytilus galloprovincialis
Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18°C was 1.5- and 3- to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4°C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of
d
-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph.
pH Signaling in
Sclerotinia sclerotiorum
: Identification of a
pacC/RIM1
Homolog
Authors: Rollins Jeffrey A. , Dickman Martin B.
Published: July 2002

Sclerotinia sclerotiorum
acidifies its ambient environment by producing oxalic acid. This production of oxalic acid during plant infection has been implicated as a primary determinant of pathogenicity in this and other phytopathogenic fungi. We found that ambient pH conditions affect multiple processes in
S. sclerotiorum
. Exposure to increasing alkaline ambient pH increased the oxalic acid accumulation independent of carbon source, sclerotial development was favored by acidic ambient pH conditions but inhibited by neutral ambient pH, and transcripts encoding the endopolygalacturonase gene
pg1
accumulated maximally under acidic culture conditions. We cloned a putative transcription factor-encoding gene,
pac1
, that may participate in a molecular signaling pathway for regulating gene expression in response to ambient pH. The three zinc finger domains of the predicted Pac1 protein are similar in sequence and organization to the zinc finger domains of the
A. nidulans
pH-responsive transcription factor PacC. The promoter of
pac1
contains eight PacC consensus binding sites, suggesting that this gene, like its homologs, is autoregulated. Consistent with this suggestion, the accumulation of
pac1
transcripts paralleled increases in ambient pH. Pac1 was determined to be a functional homolog of PacC by complementation of an
A. nidulans pacC
-null strain with
pac1
. Our results suggest that ambient pH is a regulatory cue for processes linked to pathogenicity, development, and virulence and that these processes may be under the molecular regulation of a conserved pH-dependent signaling pathway analogous to that in the nonpathogenic fungus
A. nidulans
.
Various Levels of Cross-Resistance to
Bacillus sphaericus
Strains in
Culex pipiens
(Diptera: Culicidae) Colonies Resistant to
B. sphaericus
Strain 2362
Authors: Nielsen-LeRoux Christina , Rao D. Raghunatha , Murphy Jittawadee Rodcharoen , Carron Alexandre , Mani T. R. , Hamon Sylviane , Mulla Mir S.
Published: July 2002

We studied the cross-resistance to three highly toxic
Bacillus sphaericus
strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the
Culex pipiens
complex resistant to
B. sphaericus
2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the
B. sphaericus
strains tested. Thus, among the mosquitocidal strains of
B. sphaericus
we identified a strain, IAB-59, which was toxic to several
Culex
colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of
B. sphaericus
preparations.
Identification and Characterization of the Three Chitin-Binding Domains within the Multidomain Chitinase Chi92 from
Aeromonas hydrophila
JP101
Authors: Wu Mei Li , Chuang Yin Ching , Chen Jen Pin , Chen Chin Shuh , Chang Ming Chung
Published: July 2002

The gene (
chi92
) encoding the extracellular chitinase of
Aeromonas hydrophila
JP101 has been cloned and expressed in
Escherichia coli
. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBD
CI
, and ChBD
CII
). The C-terminally repeated ChBDs, ChBD
CI
and ChBD
CII
, were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione
S
-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBD
CI
) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of
Serratia marcescens
ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBD
CI
and ChBD
CII
).
Comparison of Cellular and Biomass Specific Activities of Dominant Bacterioplankton Groups in Stratified Waters of the Celtic Sea
Authors: Zubkov Mikhail V. , Fuchs Bernhard M. , Burkill Peter H. , Amann Rudolf
Published: July 2002

A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [
35
S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of α-proteobacteria, related to
Roseobacter
spp., and a
Cytophaga-Flavobacterium
cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the α-proteobacteria exhibited high light scatter (hs) and the
Cytophaga-Flavobacterium
cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of γ-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production: different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.
Diversity of the Ring-Cleaving Dioxygenase Gene
pcaH
in a Salt Marsh Bacterial Community
Authors: Buchan Alison , Neidle Ellen L. , Moran Mary Ann
Published: July 2002

Degradation of lignin-related aromatic compounds is an important ecological process in the highly productive salt marshes of the southeastern United States, yet little is known about the mediating organisms or their catabolic pathways. Here we report the diversity of a gene encoding a key ring-cleaving enzyme of the β-ketoadipate pathway,
pcaH
, amplified from bacterial communities associated with decaying
Spartina alterniflora
, the salt marsh grass that dominates these coastal systems, as well as from enrichment cultures with aromatic substrates (
p
-hydroxybenzoate, anthranilate, vanillate, and dehydroabietate). Sequence analysis of 149
pcaH
clones revealed 85 unique sequences. Thirteen of the 53 amino acid residues compared were invariant in the PcaH proteins, suggesting that these residues have a required catalytic or structural function. Fifty-eight percent of the clones matched sequences amplified from a collection of 36 bacterial isolates obtained from seawater, marine sediments, or senescent
Spartina
. Fifty-two percent of the
pcaH
clones could be assigned to the roseobacter group, a marine lineage of the class α-
Proteobacteria
abundant in coastal ecosystems. Another 6% of the clones matched genes retrieved from isolates belonging to the genera
Acinetobacter, Bacillus
, and
Stappia
, and 42% of the clones could not be assigned to a cultured bacterium based on sequence identity. These results suggest that the diversity of the genes encoding a single step in aromatic compound degradation in the coastal marsh examined is high.
Acyl-Homoserine Lactone Production Is More Common among Plant-Associated
Pseudomonas
spp. than among Soilborne
Pseudomonas
spp
Authors: Elasri Miena , Delorme Sandrine , Lemanceau Philippe , Stewart Gordon , Laue Bridget , Glickmann Eric , Oger Phil M. , Dessaux Yves
Published: July 2002

A total of 137 soilborne and plant-associated bacterial strains belonging to different
Pseudomonas
species were tested for their ability to synthesize
N
-acyl-homoserine lactones (NAHL). Fifty-four strains synthesized NAHL. Interestingly, NAHL production appears to be more common among plant-associated than among soilborne
Pseudomonas
spp. Indeed, 40% of the analyzed
Pseudomonas syringae
strains produced NAHL which were identified most often as the short-chain NAHL,
N
-hexanoyl-
l
-homoserine lactone,
N
-(3-oxo-hexanoyl)-homoserine lactone, and
N
-(3-oxo-octanoyl)-
l
-homoserine lactone (no absolute correlation between genomospecies of
P. syringae
and their ability to produce NAHL could be found). Six strains of fluorescent pseudomonads, belonging to the species
P. chlororaphis
,
P. fluorescens
, and
P. putida
, isolated from the plant rhizosphere produced different types of NAHL. In contrast, none of the strains isolated from soil samples were shown to produce NAHL. The gene encoding the NAHL synthase in
P. syringae
pv. maculicola was isolated by complementation of an NAHL-deficient
Chromobacterium
mutant. Sequence analysis revealed the existence of a
luxI
homologue that we named
psmI
. This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to
psyI
and
ahlI
, two genes involved in NAHL production in
P. syringae
pv. tabaci and
P. syringae
pv. syringae, respectively. We identified another open reading frame that we termed
psmR
, transcribed convergently in relation to
psmI
and partly overlapping
psmI
; this gene encodes a putative LuxR regulatory protein. This gene organization, with
luxI
and
luxR
homologues facing each other and overlapping, has been found so far only in the enteric bacteria
Erwinia
and
Pantoea
and in the related species
P. syringae
pv. tabaci.
Dual Role of GdmH in Producer Immunity and Secretion of the Staphylococcal Lantibiotics Gallidermin and Epidermin
Authors: Hille Matthias , Kies Stefanie , Götz Friedrich , Peschel Andreas
Published: July 2002

The biosynthetic gene clusters of the staphylococcal lantibiotics epidermin and gallidermin are distinguished by the presence of the unique genes
epiH
and
gdmH
, respectively. They encode accessory factors for the ATP-binding cassette transporters that mediate secretion of the antimicrobial peptides. Here, we show that
gdmH
also contributes to immunity to gallidermin but not to nisin.
gdmH
alone affected susceptibility to gallidermin only moderately, but it led to a multiplication of the immunity level mediated by the
FEG
immunity genes when cloned together with the
gdmT
gene, suggesting a synergistic activity of the H and FEG systems.
gdmH
-related genes were identified in the genomes of several bacteria, indicating an involvement in further cellular functions.
Genomic Relatedness within Five Common Finnish
Campylobacter jejuni
Pulsed-Field Gel Electrophoresis Genotypes Studied by Amplified Fragment Length Polymorphism Analysis, Ribotyping, and Serotyping
Authors: Hänninen Marja-Liisa , Perko-Mäkelä Päivikki , Rautelin Hilpi , Duim Birgitta , Wagenaar Jaap A.
Published: July 2002

Thirty-five Finnish
Campylobacter jejuni
strains with five
Sma
I/
Sac
II pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns,
Hae
III ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with
Hae
III were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish
C. jejuni
genotypes form genetic lineages which colonize both humans and chickens.
Effect of Exogenous Siderophores on Iron Uptake Activity of Marine Bacteria under Iron-Limited Conditions
Authors: Guan Le Luo , Kanoh Kaneo , Kamino Kei
Published: July 2002

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore,
N
,
N
′-bis (2,3-dihydroxybenzoyl)-
O
-serylserine from a
Vibrio
sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using
59
Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.
Phylogenetic Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Deep-Sea Microorganisms
Authors: Elsaied Hosam , Naganuma Takeshi
Published: July 2002

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel,
Bathymodiolus
sp., and the seep vestimentiferan tubeworm,
Lamellibrachia
sp. The RuBisCO genes that encode both form I and form II large subunits (
cbbL
and
cbbM
) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50
cbbL
clones and 50
cbbM
clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total
cbbL
clones, and a total of 24 OTUs were recorded from the 250 total
cbbM
clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the
Thiobacillus
-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.
Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing
Pseudomonas
spp.: Characterization of Superior Root-Colonizing
P. fluorescens
Strain Q8r1-96
Authors: Raaijmakers Jos M. , Weller David M.
Published: July 2002

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing
Pseudomonas
spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing
Pseudomonas
spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by
P. fluorescens
Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed
−1
or soil
−1
) to establish high rhizosphere population densities (10
7
CFU g of root
−1
) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing
P. fluorescens
strains. Q8r1-96 maintained a rhizosphere population density of approximately 10
5
CFU g of root
−1
after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.
Strategy for Manipulation of Cheese Flora Using Combinations of Lacticin 3147-Producing and -Resistant Cultures
Authors: Ryan Máire P. , Ross R. Paul , Hill Colin
Published: July 2002

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A
Lactobacillus paracasei
subsp.
paracasei
strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either
Lactococcus lactis
DPC3147 (a natural producer) or
L. lactis
DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10
7
CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.