bluuebunny/crossref_metadata_2025 huggingface dataset:
Salinity Responses of Benthic Microbial Communities in a Solar Saltern (Eilat, Israel)
Authors: Sørensen Ketil Bernt , Canfield Donald E. , Oren Aharon
Published: March 2004

The salinity responses of cyanobacteria, anoxygenic phototrophs, sulfate reducers, and methanogens from the laminated endoevaporitic community in the solar salterns of Eilat, Israel, were studied in situ with oxygen microelectrodes and in the laboratory in slurries. The optimum salinity for the sulfate reduction rate in sediment slurries was between 100 and 120‰, and sulfate reduction was strongly inhibited at an in situ salinity of 215‰. Nevertheless, sulfate reduction was an important respiratory process in the crust, and reoxidation of formed sulfide accounted for a major part of the oxygen budget. Methanogens were well adapted to the in situ salinity but contributed little to the anaerobic mineralization in the crust. In slurries with a salinity of 180‰ or less, methanogens were inhibited by increased activity of sulfate-reducing bacteria. Unicellular and filamentous cyanobacteria metabolized at near-optimum rates at the in situ salinity, whereas the optimum salinity for anoxygenic phototrophs was between 100 and 120‰.
Identification and Isolation of Anaerobic, Syntrophic Phthalate Isomer-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephthalate Manufacturing
Authors: Qiu Yan-Ling , Sekiguchi Yuji , Imachi Hiroyuki , Kamagata Yoichi , Tseng I-Cheng , Cheng Sheng-Shung , Ohashi Akiyoshi , Harada Hideki
Published: March 2004

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37°C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group ‘Desulfotomaculumlineage I' or a clone cluster (group TA) in the class delta-Proteobacteria. Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogenMethanospirillum hungateiconverted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group ‘Desulfotomaculumlineage I', but it was only distantly related to other known species.
Direct Observation of Microbial Inhibition of Calcite Dissolution
Authors: Lüttge Andreas , Conrad Pamela G.
Published: March 2004

Vertical scanning interferometry (VSI) provides a method for quantification of surface topography at the angstrom to nanometer level. Time-dependent VSI measurements can be used to study the surface-normal retreat across crystal and other solid surfaces during dissolution or corrosion processes. Therefore, VSI can be used to directly and nondestructively measure mineral dissolution rates with high precision. We have used this method to compare the abiotic dissolution behavior of a representative calcite (CaCO
3
) cleavage face with that observed upon addition of an environmental microbe,
Shewanella oneidensis
MR-1, to the crystal surface. From our direct observations, we have concluded that the presence of the microbes results in a significant inhibition of the rate of calcite dissolution. This inhibition appears to be a 2nd-order effect that is related to the formation of etch pits. The opening of etch pits was greatly inhibited in the presence of added bacteria, suggesting that the bacterial cells exert their effect by inhibiting the formation of etch pits at high-energy sites at the crystal surface caused by lattice defects, e.g., screw or point dislocations. The experimental methodology thus provides a nondestructive, directly quantifiable, and easily visualized view of the interactions of microbes and minerals during weathering (or corrosion) processes or during mineral precipitation.
Phage Community Dynamics in Hot Springs
Authors: Breitbart Mya , Wegley Linda , Leeds Steven , Schoenfeld Tom , Rohwer Forest
Published: March 2004

In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments.
Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization
Authors: Kindaichi Tomonori , Ito Tsukasa , Okabe Satoshi
Published: March 2004

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4+as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the classProteobacteria(α-Proteobacteria), 23%; γ-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%;Cytophaga-Flavobacterium-Bacteroides(CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. α- and γ-Proteobacteriadominated the utilization of [14C]acetic acid and14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consumingN-acetyl-d-[1-14C]glucosamine (NAG). The GNSB accounted for 9% of the14C-amino acid-consuming bacteria and 27% of the [14C]NAG-consuming bacteria but did not utilize [14C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.
Rapid Detection and Enumeration of
Legionella pneumophila
in Hot Water Systems by Solid-Phase Cytometry
Authors: Aurell Helena , Catala Philippe , Farge Pierre , Wallet France , Le Brun Matthieu , Helbig Jürgen H. , Jarraud Sophie , Lebaron Philippe
Published: March 2004

A new method for the rapid and sensitive detection of
Legionella pneumophila
in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of
L. pneumophila
serogroup 1 and
L. pneumophila
serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-
Legionella
strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of
L. pneumophila
were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for
L. pneumophila
screening in hot water systems.
Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine
Roseobacter
Lineage
Authors: Buchan Alison , Neidle Ellen L. , Moran Mary Ann
Published: March 2004

Members of the
Roseobacter
lineage, an ecologically important marine clade within the class α-
Proteobacteria
, harbor genes for the protocatechuate branch of the β-ketoadipate pathway, a major catabolic route for lignin-related aromatic compounds. The genes of this pathway are typically clustered, although gene order varies among organisms. Here we characterize genes linked to
pcaH
and
-G
, which encode protocatechuate 3,4-dioxygenase, in eight closely related members of the
Roseobacter
lineage (pairwise 16S rRNA gene sequence identities, 92 to 99%). Sequence analysis of genomic fragments revealed five unique
pca
gene arrangements. Identical gene organization was found for isolates demonstrating species-level identity (i.e., >99% 16S rRNA gene similarity). In one isolate, six functionally related genes were clustered:
pcaQ
,
pobA
,
pcaD
,
pcaC
,
pcaH
, and
pcaG
. The remaining seven isolates lacked at least one of these genes in their clusters, although the relative order of the remaining genes was preserved. Three genes (
pcaC
, -
H
, and -
G
) were physically linked in all isolates. A highly conserved open reading frame (ORF) was found immediately downstream of
pcaG
in all eight isolates. Reverse transcription-PCR analysis of RNA from one isolate,
Silicibacter pomeroyi
DSS-3, provides evidence that this ORF is coexpressed with upstream
pca
genes. The absence of this ORF in similar bacterial
pca
gene clusters from diverse microbes suggests a niche-specific role for its protein product in
Roseobacter
group members. Collectively, these comparisons of bacterial
pca
gene organization illuminate a complex evolutionary history and underscore the widespread ecological importance of the encoded β-ketoadipate pathway.
pbp2229
-Mediated Nisin Resistance Mechanism in
Listeria monocytogenes
Confers Cross-Protection to Class IIa Bacteriocins and Affects Virulence Gene Expression
Authors: Gravesen Anne , Kallipolitis Birgitte , Holmstrøm Kim , Høiby Poul Erik , Ramnath Manilduth , Knøchel Susanne
Published: March 2004

It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes,
pbp2229
,
hpk1021
, and
lmo2487
, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively. In the present work, we determined the direct role of the three genes in nisin resistance. Interruption of
pbp2229
and
hpk1021
eliminated the nisin resistance phenotype. Interruption of
hpk1021
additionally abolished the increase in
pbp2229
expression. The results indicate that this nisin resistance mechanism is caused directly by the increase in
pbp2229
expression, which in turn is brought about by the increase in
hpk1021
expression. We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms. The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (
prfA
-regulated as well as
prfA
-independent genes). Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development.
Application of a Vital Fluorescent Staining Method for Simultaneous, Near-Real-Time Concentration Monitoring of Two Bacterial Strains in an Atlantic Coastal Plain Aquifer in Oyster, Virginia
Authors: Fuller Mark E. , Mailloux Brian J. , Streger Sheryl H. , Hall James A. , Zhang Pengfei , Kovacik William P. , Vainberg Simon , Johnson William P. , Onstott Tullis C. , DeFlaun Mary F.
Published: March 2004

Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001.
Comamonas
sp. strain DA001 and
Acidovorax
sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.
Possible Association of GroES and Antigen 85 Proteins with Heat Resistance ofMycobacterium paratuberculosis
Authors: Sung Nackmoon , Takayama Kuni , Collins Michael T.
Published: March 2004

Conflicting reports on the heat resistance ofMycobacterium paratuberculosisprompted an examination of the effect of culture medium on this property of the organism.M. paratuberculosiswas cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0.M. paratuberculosisgrown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids ofM. paratuberculosiswere evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance ofM. paratuberculosis. The decimal reduction times at 65°C (D65°Cvalues; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) forM. paratuberculosisstrains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P< 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), theD65°Cvalue was significantly lower than that for the organism grown in 7H9-OADC medium (P =0.022) but higher than that when it was cultured in WR-GD medium (P =0.005). Proteomic analysis by 2-DE of soluble proteins extracted fromM. paratuberculosisgrown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance ofM. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression ofM. paratuberculosisand emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.
Molecular Evidence for the Evolution of Metal Homeostasis Genes by Lateral Gene Transfer in Bacteria from the Deep Terrestrial Subsurface
Authors: Coombs J. M. , Barkay T.
Published: March 2004

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P
IB
-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P
IB
-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the β subclass of the
Proteobacteria
(β-proteobacterium) that may have predated the diversification of the genus
Pseudomonas
. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of β- and γ-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P
IB
-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.
Temporal Shedding Patterns and Virulence Factors ofEscherichia coliSerogroups O26, O103, O111, O145, and O157 in a Cohort of Beef Calves and Their Dams
Authors: Pearce M. C. , Jenkins C. , Vali L. , Smith A. W. , Knight H. I. , Cheasty T. , Smith H. R. , Gunn G. J. , Woolhouse M. E. J. , Amyes S. G. B. , Frankel G.
Published: March 2004

This study investigated the shedding ofEscherichia coliO26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays.E. coliO26 was shed by 94% of calves. Over 90% ofE. coliO26 isolates carried thevtx1,eae, andehlgenes, 6.5% carriedvtx1andvtx2, and one isolate carriedvtx2only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates.E. coliO103 was shed by 51% of calves. Forty-eight percent ofE. coliO103 isolates carriedeaeandehl, 2% carriedvtx2, and none carriedvtx1. Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding ofE. coliO145 and O157 was rare. All serogroup O145 isolates carriedeae, but none carriedvtx1orvtx2. All but one serogroup O157 isolate carriedvtx2,eae, andehl. E. coliO111 was not detected. In most calves, the temporal pattern ofE. coliO26 and O103 shedding was random.E. coliO26 was detected in three times as many samples asE. coliO103, and the rate at which calves began sheddingE. coliO26 for the first time was five times greater than that forE. coliO103. ForE. coliO26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. ForE. coliO26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.
Virucidal Efficacy of Glutaraldehyde against Enteroviruses Is Related to the Location of Lysine Residues in Exposed Structures of the VP1 Capsid Protein
Authors: Chambon Martine , Archimbaud Christine , Bailly Jean-Luc , Gourgand Jeanne-Marie , Charbonné Françoise , Peigue-Lafeuille Hélène
Published: March 2004

Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.
Microbial Diversity and Heterogeneity in Sandy Subsurface Soils
Authors: Zhou Jizhong , Xia Beicheng , Huang Heshu , Palumbo Anthony V. , Tiedje James M.
Published: March 2004

Microbial community diversity and heterogeneity in saturated and unsaturated subsurface soils from Abbott's Pit in Virginia (1.57, 3.25, and 4.05 m below surface) and Dover Air Force Base in Delaware (6.00 and 7.50 m below surface) were analyzed using a culture-independent small-subunit (SSU) rRNA gene (rDNA)-based cloning approach. Four to six dominant operational taxonomic units (OTUs) were identified in 33 to 100 unique SSU rDNA clones (constituting about 40 to 50% of the total number of SSU rDNA clones in the clone library) from the saturated subsurface samples, whereas no dominant OTUs were observed in the unsaturated subsurface sample. Less than 10% of the clones among samples from different depths at the same location were identical, and the proportion of overlapping OTUs was lower for the samples that were vertically far apart than for adjacent samples. In addition, no OTUs were shared between the Abbott's Pit and Dover samples. The majority of the clones (80%) had sequences that were less than 5% different from those in the current databases. Phylogenetic analysis indicated that most of the bacterial clones were affiliated with members of the
Proteobacteria
family (90%), gram-positive bacteria (3%), and members of the
Acidobacteria
family (3%). Principal component analysis revealed that samples from different geographic locations were well separated and that samples from the same location were closely grouped together. In addition, the nonsaturated subsurface samples from Abbott's Pit clustered together and were well separated from the saturated subsurface soil sample. Finally, the overall diversity of the subsurface samples was much lower than that of the corresponding surface soil samples.
Antisense RNA Targeting of Primase Interferes with Bacteriophage Replication inStreptococcus thermophilus
Authors: Sturino Joseph M. , Klaenhammer Todd R.
Published: March 2004

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved inStreptococcus thermophilusbacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) fromS. thermophilusphage κ3 provided significant protection from κ3 and two other Sfi21-type phages. Expression ofpri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of κ3 to 3 × 10−3and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 × 10−3. The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5′ region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression ofpri3.10-ASresulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection inS. thermophilus.
Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A
Authors: Hickey Rita M. , Ross R. Paul , Hill Colin
Published: March 2004

This study concerns the exploitation of the lytic enzyme enterolysin A (EntL
)
, produced by
Enterococcus faecalis
strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some
E. faecalis
strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of
entL
, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems.
Elucidation of the Antibacterial Mechanism of the
Curvularia
Haloperoxidase System by DNA Microarray Profiling
Authors: Hansen Eva H. , Schembri Mark A. , Klemm Per , Schäfer Thomas , Molin Søren , Gram Lone
Published: March 2004

A novel antimicrobial enzyme system, the
Curvularia
haloperoxidase system, was examined with the aim of elucidating its mechanism of antibacterial action.
Escherichia coli
strain MG1655 was stressed with sublethal concentrations of the enzyme system, causing a temporary arrest of growth. The expression of genes altered upon exposure to the
Curvularia
haloperoxidase system was analyzed by using DNA microarrays. Only a limited number of genes were involved in the response to the
Curvularia
haloperoxidase system. Among the induced genes were the
ibpA
and
ibpB
genes encoding small heat shock proteins, a gene cluster of six genes (b0301-b0306) of unknown function, and finally,
cpxP
, a member of the Cpx pathway. Knockout mutants were constructed with deletions in b0301-b0306,
cpxP
, and
cpxARP
, respectively. Only the mutant lacking
cpxARP
was significantly more sensitive to the enzyme system than was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one of the specific responses of
E. coli
—namely, the Cpx pathway, which is important for managing the stress response from the
Curvularia
haloperoxidase system.
Positive Autoregulation and Signaling Properties of Pyoluteorin, an Antibiotic Produced by the Biological Control Organism
Pseudomonas fluorescens
Pf-5
Authors: Brodhagen Marion , Henkels Marcella D. , Loper Joyce E.
Published: March 2004

Pseudomonas fluorescens
Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (
inaZ
) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.
Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray
Authors: Jenkins Bethany D. , Steward Grieg F. , Short Steven M. , Ward Bess B. , Zehr Jonathan P.
Published: March 2004

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by
nifH
. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of
nifH
amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high
nifH
sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.
Bacterial Succession in a Petroleum Land Treatment Unit
Authors: Kaplan Christopher W. , Kitts Christopher L.
Published: March 2004

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C
10
to C
32
range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with
Flavobacterium
and
Pseudomonas
were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while
Flavobacterium
and
Pseudomonas
phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.
Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (
Triticum aestivum
L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones
Authors: Conn Vanessa M. , Franco Christopher M. M.
Published: March 2004

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being
Mycobacterium
(21.02%) in Swedes Flat,
Streptomyces
(14.35%) in Red Loam, and
Kitasatospora
(15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of
Mycobacterium
and
Streptomyces
species.
Sensitivity of
Escherichia coli
O157:H7 to Commercially Available Alkaline Cleaners and Subsequent Resistance to Heat and Sanitizers
Authors: Sharma Manan , Beuchat Larry R.
Published: March 2004

The effects of seven commercially available alkaline cleaners used in the food processing industry, 0.025 M NaOH, and 0.025 M KOH on viability of wild-type (EDL 933) and
rpoS
-deficient (FRIK 816-3) strains of
Escherichia coli
O157:H7 in logarithmic and stationary phases of growth were determined. Cells were treated at 4 or 23°C for 2, 10, or 30 min. Cleaners 2, 4, 6, and 7, which contained hypochlorite and <11% NaOH and/or KOH (pH 11.2 to 11.7), killed significantly higher numbers of cells than treatment with cleaner 3, containing sodium metasilicate (pH 11.4) and <10% KOH, and cleaner 5, containing ethylene glycol monobutyl ether (pH 10.4). There were no differences in the sensitivities of logarithmic and stationary-phase cells to the alkaline cleaners. Treatment with KOH or NaOH (pH 12.2) was not as effective as four out of seven commercial cleaners in killing
E. coli
O157:H7, indicating that chlorine and other cleaner components have bactericidal activity at high pH. Stationary-phase cells of strain EDL 933 that had been exposed to cleaner 7 at 4 or 23°C and strain FRIK 816-3 exposed to cleaner 7 at 23°C had significantly higher
D
55°C
(decimal reduction time, minutes at 55°C) values than control cells or cells exposed to cleaner 5, indicating that exposure to cleaner 7 confers cross-protection to heat. Cells of EDL 933 treated with cleaner 7 at 12°C showed significantly higher
D
55°C
values than cells of FRIK 816-3, indicating that
rpoS
may play a role in cross-protection. Stationary-phase cells treated with cleaner 5 or cleaner 7 at 4 or 12°C were not cross-protected against subsequent exposure to sanitizers containing quaternary ammonium compounds or sodium hypochlorite, or to cetylpyridinium chloride and benzalkonium chloride.
Construction of Chimeric Catechol 2,3-Dioxygenase Exhibiting Improved Activity against the Suicide Inhibitor 4-Methylcatechol
Authors: Okuta Akiko , Ohnishi Kouhei , Harayama Shigeaki
Published: March 2004

Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (
xylE
or
nahH
) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into
Pseudomonas putida
possessing some of the toluene catabolic genes (
xylXYZLGFJQKJI
). When a C23O gene (e.g.,
xylE
) is introduced into this strain, the transformants cannot generally grow on
p
-toluate because 4-methylcatechol, a metabolite of
p
-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on
p
-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on
p
-toluate.
Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling
Authors: Sliwinski Marek K. , Goodman Robert M.
Published: March 2004

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division
Crenarchaeota
(domain
Archaea
). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [
P
< 0.0001] and least-significant-difference analysis [α = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.
Comparison of Crenarchaeal Consortia Inhabiting the Rhizosphere of Diverse Terrestrial Plants with Those in Bulk Soil in Native Environments
Authors: Sliwinski Marek K. , Goodman Robert M.
Published: March 2004

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T
2
test,
P
< 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.
Detection of
Cryptosporidium parvum
in Soil Extracts
Authors: Walker Mark , Redelman Douglas
Published: March 2004

Epifluorescent microscopy and flow cytometry were used in different combinations with fluorescein isothiocyanate-labeled immunoglobulins M and G3 to estimate the numbers of
Cryptosporidium parvum
oocysts in soil extracts containing 10 to 10,017 oocysts/ml. No combination had a systematic effect on accuracy or precision. Background debris may have produced overestimates at low oocyst concentrations when flow cytometry was used.
Detection of
Escherichia coli
Serogroups O26 and O113 by PCR Amplification of the
wzx
and
wzy
Genes
Authors: DebRoy Chitrita , Roberts Elisabeth , Kundrat James , Davis Michael A. , Briggs Connie E. , Fratamico Pina M.
Published: March 2004

PCR-based assays for detecting enterohemorrhagic
Escherichia coli
serogroups O26 and O113 were developed by targeting the
wzx
(O-antigen flippase) and the
wzy
(O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113
E. coli
serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as ≤10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing
E. coli
O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.
Inactivation of
Vibrio parahaemolyticus
in Effluent Seawater by Alternating-Current Treatment
Authors: Park Jong-Chul , Lee Min Sub , Han Dong-Wook , Lee Dong Hee , Park Bong Joo , Lee In-Seop , Uzawa Masakazu , Aihara Maki , Takatori Kosuke
Published: March 2004

Vibrio parahaemolyticus
, the cause of gastroenteritis in humans, was inactivated by alternating low-amperage electricity. In this study, the application of alternating low-amperage electric treatment to effluent seawater was investigated for the large-scale disinfection of seawater. This method was able to overcome the problem of chlorine generation that results from treatment with continuous direct current. In conclusion, our results showed that alternating-current treatment inactivates
V. parahaemolyticus
in effluent seawater while minimizing the generation of chlorine and that this alternating-current treatment is therefore suitable for practical industrial applications.