bluuebunny/crossref_metadata_2025_split huggingface dataset:
Putative Transposases Conserved in
Exiguobacterium
Isolates from Ancient Siberian Permafrost and from Contemporary Surface Habitats
Authors: Vishnivetskaya Tatiana A. , Kathariou Sophia 
Published: November 2005

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.
Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community
Authors: Ferris M. J. , Sheehan K. B. , Kühl M. , Cooksey K. , Wigglesworth-Cooksey B. , Harvey R. , Henson J. M. 
Published: November 2005

ABSTRACTUnicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. ACyanidioschyzon merolaesequence was predominant at temperatures of ≥49°C. AChlorella protothecoidesvar.acidicolasequence and aParadoxia multisita-like sequence were predominant at temperatures of ≤39°C.
Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation
Authors: Senko John M. , Dewers Thomas A. , Krumholz Lee R. 
Published: November 2005

ABSTRACT

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to
Klebsiella oxytoca.
FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)
sol
] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)
sol
yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)
sol
and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.
Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of
Pasteurellaceae
Authors: Briggs Robert E. , Tatum Fred M. 
Published: November 2005

ABSTRACT

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from
Mannheimia hemolytica
serotype 1. Three TS plasmids which failed to replicate at or above 42°C in
M. hemolytica
but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in
Pasteurella multocida
and
Haemophilus somnus
as well. To consistently transform
H. somnus
with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.
Construction of In-Frame
aroA
Deletion Mutants of
Mannheimia haemolytica
,
Pasteurella multocida
, and
Haemophilus somnus
by Using a New Temperature-Sensitive Plasmid
Authors: Tatum Fred M. , Briggs Robert E. 
Published: November 2005

ABSTRACT

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of
Mannheimia hemolytica
and used to engineer in-frame
aroA
deletion mutants of
Mannheimia hemolytica
,
Pasteurella multocida
, and
Haemophilus somnus
. TS replacement plasmids carrying in-frame
aroA
deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of
Pasteurellaceae
species.