bluuebunny/crossref_metadata_2025 huggingface dataset:
CorA Affects Tolerance of
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress
Authors: Sermon Jan , Wevers Eva M.-R. P. , Jansen Leentje , De Spiegeleer Philipp , Vanoirbeek Kristof , Aertsen Abram , Michiels Chris W.
Published: November 2005

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in
Escherichia coli
MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of
E. coli
MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (
corA
) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the
corA
mutant.
Salmonella enterica
serovar Typhimurium
corA
had a similar phenotype. Although
corA
encodes an Mg
2+
transporter and at least three other inducible open reading frames belonged to the Mg
2+
regulon, repression of the Mg stimulon by Mg
2+
did not change the lactoperoxidase sensitivity of either the wild-type or
corA
mutant. Prior exposure to 0.3 mM Ni
2+
, which is also transported by CorA, strongly sensitized MG1655 but not the
corA
mutant to the lactoperoxidase system. Furthermore, this Ni
2+
-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of
E. coli
by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.
Lactobacillus paracasei
subsp.
paracasei
8700:2 Degrades Inulin-Type Fructans Exhibiting Different Degrees of Polymerization
Authors: Makras Lefteris , Van Acker Gerald , De Vuyst Luc
Published: November 2005

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate
Lactobacillus acidophilus
IBB 801 degraded only oligofructose. The human isolate
Lactobacillus paracasei
subsp.
paracasei
8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When
L. paracasei
subsp.
paracasei
8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.
Isolation and Characterization of a Novel Antialgal Allelochemical fromPhragmites communis
Authors: Li Feng-Min , Hu Hong-Ying
Published: November 2005

Antialgal allelochemicals were isolated fromPhragmites communisTris. The isolated allelopathic fraction showed strong inhibition activity on the growth ofChlorella pyrenoidosaandMicrocystis aeruginosabut had no inhibition onChlorella vulgaris.The 50% effective concentrations (EC50) of the allelopathic fractions onC. pyrenoidosaandM. aeruginosawere 0.49 and 0.79 mg/liter, respectively. The allelopathic activity of the fraction was species-specific. The isolated allelopathic fraction caused metal ion leakage from algal cells. The fraction decreased the activities of antioxidant enzymes, such as superoxide dismutase and peroxidase. The addition of the isolated fraction increased the concentration of unsaturated lipid fatty acids in cell membrane ofC. pyrenoidosaandM. aeruginosa. This caused a change in plasma membrane integrity and the leakage of ions in the protoplast. The allelopathic compound was identified by nuclear magnetic resonance and gas chromatography-mass spectrometry as ethyl 2-methylacetoacetate. Synthesized ethyl 2-methylacetoacetate also showed allelopathic activity onC. pyrenoidosaandM. aeruginosa. The EC50of synthesized ethyl 2-methylacetoacetate onC. pyrenoidosaandM. aeruginosawere 0.49 and 0.65 mg/liter, respectively.
Bacteriophage Therapy To Reduce
Campylobacter jejuni
Colonization of Broiler Chickens
Authors: Loc Carrillo C. , Atterbury R. J. , El-Shibiny A. , Connerton P. L. , Dillon E. , Scott A. , Connerton I. F.
Published: November 2005

Colonization of broiler chickens by the enteric pathogen
Campylobacter jejuni
is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of
Campylobacter
colonization of broiler chickens were established by using low-passage
C. jejuni
isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50
Campylobacter
isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the
C. jejuni
broiler isolates. Phage treatment of
C. jejuni
-colonized birds resulted in
Campylobacter
counts falling between 0.5 and 5 log
10
CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-
Campylobacter
combination, the dose of phage applied, and the time elapsed after administration.
Campylobacter
s resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.
Prediction of the Adaptability of
Pseudomonas putida
DOT-T1E to a Second Phase of a Solvent for Economically Sound Two-Phase Biotransformations
Authors: Neumann Grit , Kabelitz Nadja , Zehnsdorf Andreas , Miltner Anja , Lippold Holger , Meyer Daniel , Schmid Andreas , Heipieper Hermann J.
Published: November 2005

The strain
Pseudomonas putida
DOT-T1E was tested for its ability to tolerate second phases of different alkanols for their use as solvents in two-liquid-phase biotransformations. Although 1-decanol showed an about 10-fold higher toxicity to the cells than 1-octanol, the cells were able to adapt completely to 1-decanol only and could not be adapted in order to grow stably in the presence of a second phase of 1-octanol. The main explanation for this observation can be seen in the higher water and membrane solubility of 1-octanol. The hydrophobicity (log P) of a substance correlates with a certain partitioning of that compound into the membrane. Combining the log P value with the water solubility, the maximum membrane concentration of a compound can be calculated. With this simple calculation, it is possible to predict the property of an organic chemical for its potential applicability as a solvent for two-liquid-phase biotransformations with solvent-tolerant
P. putida
strains. Only compounds that show a maximum membrane concentration of less than 400 mM, such as 1-decanol, seem to be tolerated by these bacterial strains when applied in supersaturating concentrations to the medium. Taking into consideration that a solvent for a two-liquid-phase system should possess partitioning properties for potential substrates and products of a fine chemical synthesis, it can be seen that 1-decanol is a suitable solvent for such biotransformation processes. This was also demonstrated in shake cultures, where increasing amounts of a second phase of 1-decanol led to bacteria tolerating higher concentrations of the model substrate 3-nitrotoluene. Transferring this example to a 5-liter-scale bioreactor with 10% (vol/vol) 1-decanol, the amount of 3-nitrotoluene tolerated by the cells is up to 200-fold higher than in pure aqueous medium. The system demonstrates the usefulness of two-phase biotransformations utilizing solvent-tolerant bacteria.
Comparison of Minisatellite Polymorphisms in the
Bacillus cereus
Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to
Bacillus anthracis
Authors: Valjevac Samina , Hilaire Valérie , Lisanti Olivier , Ramisse Françoise , Hernandez Eric , Cavallo Jean-Didier , Pourcel Christine , Vergnaud Gilles
Published: November 2005

Polymorphism of five tandem repeats that are monomorphic in
Bacillus anthracis
was investigated in 230 isolates of the
B. cereus
group and in 5 sequenced
B. cereus
genomes in search for markers allowing identification of
B. cereus
and
B. thuringiensis
strains most closely related to
B. anthracis
. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from
B. anthracis
and was also proposed to be closest to
B. anthracis
by MLVA analysis. However, this strain remains separated from
B. anthracis
by a number of significant genetic events observed in
B. anthracis
, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of
B. cereus
and
B. thuringiensis
strains closely related to
B. anthracis
. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the
B. cereus
complex, with modest equipment and at a low cost, to eventually fill the gap with
B. anthracis
and better understand the origin and making of this dangerous pathogen.
Insights into
Actinobacillus succinogenes
Fermentative Metabolism in a Chemically Defined Growth Medium
Authors: McKinlay James B. , Zeikus J. Gregory , Vieille Claire
Published: November 2005

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing
Actinobacillus succinogenes
. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH
4
Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids.
A. succinogenes
growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO
3
concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The
A. succinogenes
growth rate was 1.3 to 1.4 times higher at an NaHCO
3
concentration of 25 mM than at any other NaHCO
3
concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the
A. succinogenes
metabolic map, the reasons for
A. succinogenes
glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or α-ketoglutarate. The inability to synthesize α-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in
A. succinogenes
.
Identification ofShewanella balticaas the Most Important H2S-Producing Species during Iced Storage of Danish Marine Fish
Authors: Vogel Birte Fonnesbech , Venkateswaran Kasthuri , Satomi Masataka , Gram Lone
Published: November 2005

Shewanella putrefacienshas been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropicShewanellahave been reclassified during recent years, and the purpose of the present study was to determine whether any of the newShewanellaspecies are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified asShewanellaspecies by phenotypic tests. DifferentShewanellaspecies were present on newly caught fish. During the warm summer months the mesophilic human pathogenicS. algaedominated the H2S-producing bacterial population. After iced storage, a shift in theShewanellaspecies was found, and most of the H2S-producing strains were identified asS. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genusShewanellalevel and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains ofS. putrefaciens,S. oneidensis,S. colwelliana, andS. affinis, but the high G+C% group clustered close toS. colwellianaby 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species.S. baltica, and the low G+C% group ofShewanellaspp. strains grew well in cod juice at 0°C, but three high G+CShewanellaspp. were unable to grow at 0°C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified asS. baltica.
Impact of Bt Corn on Rhizospheric and Soil Eubacterial Communities and on Beneficial Mycorrhizal Symbiosis in Experimental Microcosms
Authors: Castaldini M. , Turrini A. , Sbrana C. , Benedetti A. , Marchionni M. , Mocali S. , Fabiani A. , Landi S. , Santomassimo F. , Pietrangeli B. , Nuti M. P. , Miclaus N. , Giovannetti M.
Published: November 2005

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin fromBacillus thuringiensis, encoded by the truncatedCry1Abgene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiontGlomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.
Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases
Authors: Jesenská Andrea , Pavlová Martina , Strouhal Michal , Chaloupková Radka , Těšínská Iva , Monincová Marta , Prokop Zbyněk , Bartoš Milan , Pavlík Ivo , Rychlík Ivan , Möbius Petra , Nagata Yuji , Damborský Jiři
Published: November 2005

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes ofMycobacterium tuberculosisandM. boviscontain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genesdmbAanddmbBfromM. bovis5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of theM. tuberculosiscomplex, includingM. bovis,M. bovisBCG,M. africanum,M. caprae,M. microti, andM. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed inEscherichia coliand purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.
Abundance, Activity, and Community Structure of Pelagic Methane-Oxidizing Bacteria in Temperate Lakes
Authors: Sundh Ingvar , Bastviken David , Tranvik Lars J.
Published: November 2005

The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a
14
CH
4
transformation method. The fatty acid analyses indicated that type I MOB most similar to species of
Methylomonas
,
Methylomicrobium
, and
Methylosarcina
made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (
V
) ranged from 0.001 to 2.77 mg CH
4
-C mg
−1
C day
−1
and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH
4
.
Detection of Infectious
Cryptosporidium
Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples
Authors: Schets F. M. , Engels G. B. , During M. , de Roda Husman A. M.
Published: November 2005

In the past few years many waterborne outbreaks related to
Cryptosporidium
have been described. Current methods for detection of
Cryptosporidium
in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with
Cryptosporidium
this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with
Cryptosporidium
oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring
Cryptosporidium
oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with
Cryptosporidium
. For accurate risk assessment, further improvement of the method for detection of
Cryptosporidium
in water is needed.
Growth and Grazing Mortality Rates of Phylogenetic Groups of Bacterioplankton in Coastal Marine Environments
Authors: Yokokawa Taichi , Nagata Toshi
Published: November 2005

Dilution culture experiments were conducted in western North Pacific coastal regions to determine growth and grazing mortality rates of bacterial phylogenetic groups (α-, β-, and γ-proteobacteria and the
Cytophaga-Flavobacter
cluster) detected by fluorescent in situ hybridization. Growth rates varied greatly (1.2- to 4.0-fold) among different groups, and they were related to environmental variables (chlorophyll
a
concentrations and temperature) in a group-specific fashion. Growth rates of α-proteobacteria, the most abundant group in all the samples examined, were generally lower than those of less abundant groups, including the
Cytophaga-Flavobacter
cluster and γ-proteobacteria. Grazing mortality rates and mean cell volumes varied little among different groups. These results provide insights into factors that affect distributions of different groups, but growth and grazing mortality alone did not fully explain bacterial community compositions at a broad phylogenetic level.
A Propionate-Inducible Expression System for Enteric Bacteria
Authors: Lee Sung Kuk , Keasling Jay D.
Published: November 2005

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in
Escherichia coli
. The pPro vectors contain the
prpBCDE
promoter (P

prpB

) responsible for expression of the propionate catabolic genes (
prpBCDE
) and
prpR
encoding the positive regulator of this promoter. The efficiency and regulatory properties of the
prpR
-P

prpB

system were measured by placing the gene encoding the green fluorescent protein (
gfp
) under the control of the inducible P

prpB

of
E. coli
. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the
prpBCDE
promoter has CAP-dependent activation, the
prpR
-P

prpB

system exhibited negligible basal expression by addition of glucose to the medium.
Common, but Complex, Mode of Resistance of
Plutella xylostella
to
Bacillus thuringiensis
Toxins Cry1Ab and Cry1Ac
Authors: Sayyed Ali H. , Gatsi Roxani , Ibiza-Palacios M. Sales , Escriche Baltasar , Wright Denis J. , Crickmore Neil
Published: November 2005

A field collected population of
Plutella xylostella
(SERD4) was selected in the laboratory with
Bacillus thuringiensis
endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.
Reciprocal Regulation of Pyoluteorin Production with Membrane Transporter Gene Expression in
Pseudomonas fluorescens
Pf-5
Authors: Brodhagen Marion , Paulsen Ian , Loper Joyce E.
Published: November 2005

Pyoluteorin is a chlorinated polyketide antibiotic secreted by the rhizosphere bacterium
Pseudomonas fluorescens
Pf-5. Genes encoding enzymes and transcriptional regulators involved in pyoluteorin production are clustered in the genome of Pf-5. Sequence analysis of genes adjacent to the known pyoluteorin biosynthetic gene cluster revealed the presence of an ABC transporter system. We disrupted two putative ABC transporter genes by inserting transcriptional fusions to an ice nucleation reporter gene. Mutations in
pltI
and
pltJ
, which are predicted to encode a membrane fusion protein and an ATP-binding cassette of the ABC transporter, respectively, greatly reduced pyoluteorin production by Pf-5. During the transition from exponential growth to stationary phase, populations of a
pltI
mutant were lower than those of a
pltI
+
strain in a culture medium containing pyoluteorin, suggesting a role for the transport system in efflux and the resistance of Pf-5 to the antibiotic. Although
pltI
or
pltJ
mutant strains displayed low pyoluteorin production, they did not accumulate proportionately more of the antibiotic intracellularly, indicating that
pltI
and
pltJ
do not encode an exclusive exporter for pyoluteorin. Transcription of the putative pyoluteorin efflux genes
pltI
and
pltJ
was enhanced by exogenous pyoluteorin. These new observations parallel an earlier finding that pyoluteorin enhances the transcription of pyoluteorin biosynthesis genes and pyoluteorin production in Pf-5. This report provides evidence of a coordination of pyoluteorin production and the transcription of genes encoding a linked transport apparatus, wherein each requires the other for optimal expression.
Photodynamic Inactivation of
Bacillus
Spores, Mediated by Phenothiazinium Dyes
Authors: Demidova Tatiana N. , Hamblin Michael R.
Published: November 2005

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that
Bacillus
spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of
Bacillus cereus
and
B. thuringiensis
are most susceptible,
B. subtilis
and
B. atrophaeus
are also killed, and
B. megaterium
is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.
Identifying Components of the NF-κB Pathway in the Beneficial
Euprymna scolopes
-
Vibrio fischeri
Light Organ Symbiosis
Authors: Goodson Michael S. , Kojadinovic Mila , Troll Joshua V. , Scheetz Todd E. , Casavant Thomas L. , Soares M. Bento , McFall-Ngai Margaret J.
Published: November 2005

The Toll/NF-κB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of
Euprymna scolopes
was used to identify members of the Toll/NF-κB pathway. Full-length transcripts were identified by using 5′ and 3′ RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-κB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-κB pathway in the initiation of the beneficial symbiosis between
E. scolopes
and
Vibrio fischeri
.
Putative Transposases Conserved in
Exiguobacterium
Isolates from Ancient Siberian Permafrost and from Contemporary Surface Habitats
Authors: Vishnivetskaya Tatiana A. , Kathariou Sophia
Published: November 2005

Gram-positive bacteria of the genus
Exiguobacterium
have been repeatedly isolated from Siberian permafrost ranging in age from 20,000 to 2 to 3 million years and have been sporadically recovered from markedly diverse habitats, including microbial mats in Lake Fryxell (Antarctic), surface water, and food-processing environments. However, there is currently no information on genomic diversity of this microorganism or on the physiological strategies that have allowed its survival under prolonged freezing in the permafrost. Analysis of the genome sequence of the most ancient available
Exiguobacterium
isolate (
Exiguobacterium
sp. strain 255-15, from 2 to 3 million-year-old Siberian permafrost) revealed numerous putative transposase sequences, primarily of the IS
200
/IS
605
, IS
30
, and IS
3
families, with four transposase families identified. Several of the transposase genes appeared to be part of insertion sequences. Southern blots with different transposase probes yielded high-resolution genomic fingerprints which differentiated the different permafrost isolates from each other and from the
Exiguobacterium
spp. type strains which have been derived from diverse surface habitats. Each of the
Exiguobacterium
sp. strain 255-15 transposases that were used as probes had highly conserved homologs in the genome of other
Exiguobacterium
strains, both from permafrost and from modern sites. These findings suggest that, prior to their entrapment in permafrost,
Exiguobacterium
isolates had acquired transposases and that conserved transposases are present in
Exiguobacterium
spp., which now can be isolated from various modern surface habitats.
Isolation of
Mycobacterium avium
subsp.
paratuberculosis
from Free-Ranging Birds and Mammals on Livestock Premises
Authors: Corn Joseph L. , Manning Elizabeth J. B. , Sreevatsan Srinand , Fischer John R.
Published: November 2005

Surveys for
Mycobacterium avium
subsp.
paratuberculosis
infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS
900
/IS
1311
PCR and mycobactin dependency characteristics.
M. avium
subsp.
paratuberculosis
was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife
M. avium
subsp.
paratuberculosis
isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where
M. avium
subsp.
paratuberculosis
recently has been eliminated or on farms free of
M. avium
subsp.
paratuberculosis
but located in the geographic vicinity of farms with infected livestock.
Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the
ema1
Cytochrome P450 Monooxygenase
Authors: Molnár István , Hill D. Steven , Zirkle Ross , Hammer Philip E. , Gross Frank , Buckel Thomas G. , Jungmann Volker , Pachlatko Johannes Paul , Ligon James M.
Published: November 2005

The cytochrome P450 monooxygenase Ema1 from
Streptomyces tubercidicus
R-922 and its homologs from closely related
Streptomyces
strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol.
71:
6968-6976, 2005). The gene for Ema1 has been expressed in
Streptomyces lividans
,
Streptomyces avermitilis
, and solvent-tolerant
Pseudomonas putida
strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of
S. lividans
strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from
Streptomyces coelicolor
and biocatalytic
Streptomyces
strains and tested in
ema1
coexpression systems to optimize the electron transport towards Ema1.
Comparison of Microbial Community Compositions of Two Subglacial Environments Reveals a Possible Role for Microbes in Chemical Weathering Processes
Authors: Skidmore Mark , Anderson Suzanne P. , Sharp Martin , Foght Julia , Lanoil Brian D.
Published: November 2005

Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the
Betaproteobacteria
, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.
Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil
Authors: Viñas Marc , Sabaté Jordi , Espuny María José , Solanas Anna M.
Published: November 2005

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(
a
)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-
Proteobacteria
group (genera
Sphingomonas
and
Azospirillum
) was the dominant group in all treatments. At later stages, the γ-
Proteobacteria
group (genus
Xanthomonas
), the α-
Proteobacteria
group (genus
Sphingomonas
), and the
Cytophaga-Flexibacter-Bacteroides
group (
Bacteroidetes
) were the dominant groups in the nonnutrient treatment, while the γ-
Proteobacteria
group (genus
Xathomonas
), the β-
Proteobacteria
group (genera
Alcaligenes
and
Achromobacter
), and the α-
Proteobacteria
group (genus
Sphingomonas
) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.
Evidence for a Functional Quorum-Sensing Type AI-1 System in the Extremophilic Bacterium
Acidithiobacillus ferrooxidans
Authors: Farah Carolina , Vera Mario , Morin Danièle , Haras Dominique , Jerez Carlos A. , Guiliani Nicolas
Published: November 2005

Acidithiobacillus ferrooxidans
is one of the main acidophilic chemolithotrophic bacteria involved in the bioleaching of metal sulfide ores. The bacterium-mineral interaction requires the development of biofilms, whose formation is regulated in many microorganisms by type AI-1 quorum sensing. Here, we report the existence and characterization of a functional type AI-1 quorum-sensing system in
A. ferrooxidans
. This microorganism produced mainly acyl-homoserine lactones (AHL) with medium and large acyl chains and different C-3 substitutions, including 3-hydroxy-C
8
-AHL, 3-hydroxy-C
10
-AHL, C
12
-AHL, 3-oxo-C
12
-AHL, 3-hydroxy-C
12
-AHL, C
14
-AHL, 3-oxo-C
14
-AHL, 3-hydroxy-C
14
-AHL, and 3-hydroxy-C
16
-AHL. A quorum-sensing genetic locus that includes two open reading frames,
afeI
and
afeR
, which have opposite orientations and code for proteins with high levels of similarity to members of the acyl synthase (I) and transcriptional regulator (R) protein families, respectively, was identified. Overexpression of AfeI in
Escherichia coli
and the associated synthesis of AHLs confirmed that AfeI is an AHL synthase. As determined by reverse transcription-PCR, the
afeI
and
afeR
genes were transcribed in
A. ferrooxidans
. The transcription levels of the
afeI
gene were higher in cells grown in sulfur and thiosulfate media than in iron-grown cells. Phosphate starvation induced an increase in the transcription levels of
afeI
which correlated with an increase in AHL levels. Two
afe
boxes which could correspond to the AfeR binding sites were identified upstream of the
afeI
gene. This is the first report of a functional type AI-1 quorum-sensing system in an acidophilic chemolithotrophic microorganism, and our results provide a very interesting opportunity to explore the control and regulation of biofilm formation during the bioleaching process.
New Findings on Evolution of Metal Homeostasis Genes: Evidence from Comparative Genome Analysis of Bacteria and Archaea
Authors: Coombs J. M. , Barkay T.
Published: November 2005

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P
IB
-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P
IB
-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P
IB
-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.
Kluyveromyces lactis LAC4
Promoter Variants That Lack Function in Bacteria but Retain Full Function in
K. lactis
Authors: Colussi Paul A. , Taron Christopher H.
Published: November 2005

The strong
LAC4
promoter (P
LAC4
) from
Kluyveromyces lactis
has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P
LAC4
to promote gene expression in
Escherichia coli
. This can interfere with the process of assembling expression constructs in
E. coli
cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P
LAC4
variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the
E. coli
Pribnow box element of bacterial promoters and that reside immediately upstream of two
E. coli
transcription initiation sites associated with P
LAC4
. Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ∼87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in
K. lactis
cells to levels comparable to wild-type P
LAC4
. We created a novel integrative expression vector (pKLAC1) containing the P
LAC4
variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in
E. coli
cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in
E. coli
prior to their expression in
K. lactis
.
Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community
Authors: Kato Souichiro , Haruta Shin , Cui Zong Jun , Ishii Masaharu , Igarashi Yasuo
Published: November 2005

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (
Clostridium straminisolvens
CSK1,
Clostridium
sp. strain FG4,
Pseudoxanthomonas
sp. strain M1-3,
Brevibacillus
sp. strain M1-5, and
Bordetella
sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (
C. straminisolvens
CSK1) and two strains of aerobic bacteria (
Pseudoxanthomonas
sp. strain M1-3 and
Brevibacillus
sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and
C. straminisolvens
CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by
Clostridium
sp. strain FG4, and growth suppression of
Bordetella
sp. strain M1-6 by
Brevibacillus
sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.