bluuebunny/crossref_metadata_2025_split huggingface dataset:
Possible Association of GroES and Antigen 85 Proteins with Heat Resistance ofMycobacterium paratuberculosis
Authors: Sung Nackmoon , Takayama Kuni , Collins Michael T. 
Published: March 2004

ABSTRACTConflicting 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

ABSTRACT

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

ABSTRACTThis 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

ABSTRACT
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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.