bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of Alkenes via Degradation of
tert
-Alkyl Ethers and Alcohols by Aquincola tertiaricarbonis L108 and Methylibium spp
Authors: Schäfer Franziska , Muzica Liudmila , Schuster Judith , Treuter Naemi , Rosell Mònica , Harms Hauke , Müller Roland H. , Rohwerder Thore
Published: July 2011

Bacterial degradation pathways of fuel oxygenates such as methyl
tert
-butyl and
tert
-amyl methyl ether (MTBE and TAME, respectively) have already been studied in some detail. However, many of the involved enzymes are still unknown, and possible side reactions have not yet been considered. In
Aquincola tertiaricarbonis
L108,
Methylibium petroleiphilum
PM1, and
Methylibium
sp. strain R8, we have now detected volatile hydrocarbons as by-products of the degradation of the
tert
-alkyl ether metabolites
tert
-butyl and
tert
-amyl alcohol (TBA and TAA, respectively). The alkene isobutene was formed only during TBA catabolism, while the beta and gamma isomers of isoamylene were produced only during TAA conversion. Both
tert
-alkyl alcohol degradation and alkene production were strictly oxygen dependent. However, the relative contribution of the dehydration reaction to total alcohol conversion increased with decreasing oxygen concentrations. In resting-cell experiments where the headspace oxygen content was adjusted to less than 2%, more than 50% of the TAA was converted to isoamylene. Isobutene formation from TBA was about 20-fold lower, reaching up to 4% alcohol turnover at low oxygen concentrations. It is likely that the putative
tert
-alkyl alcohol monooxygenase MdpJ, belonging to the Rieske nonheme mononuclear iron enzymes and found in all three strains tested, or an associated enzymatic step catalyzed the unusual elimination reaction. This was also supported by the detection of
mdpJK
genes in MTBE-degrading and isobutene-emitting enrichment cultures obtained from two treatment ponds operating at Leuna, Germany. The possible use of alkene formation as an easy-to-measure indicator of aerobic fuel oxygenate biodegradation in contaminated aquifers is discussed.
Identification and Characterization of Potential Performance-Related Gut Microbiotas in Broiler Chickens across Various Feeding Trials
Authors: Torok Valeria A. , Hughes Robert J. , Mikkelsen Lene L. , Perez-Maldonado Rider , Balding Katherine , MacAlpine Ron , Percy Nigel J. , Ophel-Keller Kathy
Published: July 2011

Three broiler feeding trials were investigated in order to identify gut bacteria consistently linked with improvements in bird performance as measured by feed efficiency. Trials were done in various geographic locations and varied in diet composition, broiler breed, and bird age. Gut microbial communities were investigated using microbial profiling. Eight common performance-linked operational taxonomic units (OTUs) were identified within both the ilea (180, 492, and 564–566) and ceca (140–142, 218–220, 284–286, 312, and 482) across trials. OTU 564–566 was associated with lower performance, while OTUs 140–142, 482, and 492 were associated with improved performance. Targeted cloning and sequencing of these eight OTUs revealed that they represented 26 bacterial species or phylotypes which clustered phylogenetically into seven groups related to
Lactobacillus
spp.,
Ruminococcaceae
,
Clostridiales
,
Gammaproteobacteria
,
Bacteroidales
,
Clostridiales
/
Lachnospiraceae
, and unclassified bacteria/clostridia. Where bacteria were identifiable to the phylum level, they belonged predominantly to the
Firmicutes
, with
Bacteroidetes
and
Proteobacteria
also identified. Some of the potential performance-related phylotypes showed high sequence identity with classified bacteria (
Lactobacillus salivarius
,
Lactobacillus aviarius
,
Lactobacillus crispatus
,
Faecalibacterium prausnitzii
,
Escherichia coli
,
Gallibacterium anatis
,
Clostridium lactatifermentans
,
Ruminococcus torques
,
Bacteroides vulgatus
, and
Alistipes finegoldii
). The 16S rRNA gene sequence information generated will allow quantitative assays to be developed which will enable elucidations of which of these phylotypes are truly performance related. This information could be used to monitor strategies to improve feed efficiency and feed formulation for optimal gut health.
Staphylococcus aureus Host Range and Human-Bovine Host Shift
Authors: Sakwinska Olga , Giddey Marlyse , Moreillon Martine , Morisset Delphine , Waldvogel Andreas , Moreillon Philippe
Published: July 2011

Staphylococcus aureus
is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant
S. aureus
(MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated
S. aureus
strains, something which has not been systematically studied previously. The genetic relatedness of 500
S. aureus
isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and
spa
typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of
S. aureus
isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.
Repeated Anaerobic Microbial Redox Cycling of Iron
Authors: Coby Aaron J. , Picardal Flynn , Shelobolina Evgenya , Xu Huifang , Roden Eric E.
Published: July 2011

Some nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifyingBetaproteobacteria(e.g.,Dechloromonas) and Fe(III)-reducingDeltaproteobacteria(Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that bothGeobacterand variousBetaproteobacteriaparticipated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.
Pulsed-Field Gel Electrophoresis and PCR Characterization of Environmental Vibrio parahaemolyticus Strains of Different Origins
Authors: Suffredini E. , Lopez-Joven C. , Maddalena L. , Croci L. , Roque A.
Published: July 2011

The present study used pulsed-field gel electrophoresis (PFGE) characterization to examine the intraspecies variability and genetic relationships among environmental isolates of
Vibrio parahaemolyticus
from different European countries. This is first study performed on environmental
V. parahaemolyticus
that included more than one European country.
Genetic and Biochemical Map for the Biosynthesis of Occidiofungin, an Antifungal Produced by Burkholderia contaminans Strain MS14
Authors: Gu Ganyu , Smith Leif , Liu Aixin , Lu Shi-En
Published: July 2011

A striking feature of
Burkholderia contaminans
strain MS14 is the production of a glycolipopeptide named occidiofungin. Occidiofungin has a broad range of antifungal activities against plant and animal pathogens. In this study, a complete covalent structure characterization and identification of the whole genomic DNA region for the occidiofungin gene (
ocf
) cluster are described. Discovery of the presence of 2,4-diaminobutyric acid and 3-chloro-β-hydroxytyrosine and elucidation of the structure of a novel C
18
fatty amino acid residue have been achieved. In addition, seven additional putative open reading frames (the genes from
ocfI
to
ocfN
[
ocfI-N
] and ORF16) were identified. Transcription of all the putative genes
ocfI-N
identified in the region except ORF16 was regulated by both
ambR1
and
ambR2
. Elucidation of the structure and the
ocf
gene cluster provides insight into the biosynthesis of occidiofungin and promotes future aims at understanding the biosynthetic machinery. This work provides new avenues for optimizing the production and synthesis of structural analogs of occidiofungin.
Quantitative Analysis of the Relative Transcript Levels of Four Chlorophenol Reductive Dehalogenase Genes in Desulfitobacterium hafniense PCP-1 Exposed to Chlorophenols
Authors: Bisaillon Ariane , Beaudet Réjean , Lépine François , Villemur Richard
Published: July 2011

Relative to those of unexposed cultures, the transcript levels of the four CprA-type reductive dehalogenase genes (
cprA2
,
cprA3
,
cprA4
, and
cprA5
) in
Desulfitobacterium hafniense
PCP-1 were measured in cultures exposed to chlorophenols. In 2,4,6-trichlorophenol-amended cultures,
cprA2
and
cprA3
were upregulated, as was
cprA5
, but concomitantly with the appearance of 2,4-dichlorophenol (DCP). In 3,5-DCP-amended cultures, only
cprA5
was upregulated. In pentachlorophenol-amended cultures grown for 12 h,
cprA2
and
cprA3
were upregulated but not
cprA5. cprA4
was not upregulated significantly in cultures containing any tested chlorophenols.
Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry
Authors: Parthuisot N. , Binet M. , Touron-Bodilis A. , Pougnard C. , Lebaron P. , Baudart J.
Published: July 2011

A new method was developed for the rapid and sensitive detection of viable
Legionella pneumophila
. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable
L. pneumophila
cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter
−1
, and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable
L. pneumophila
cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10
3
viable but nonculturable
L. pneumophila
cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable
L. pneumophila
in both man-made water systems and environmental water samples.
Novel Multiplex Single Nucleotide Polymorphism-Based Method for Identifying Epidemic Clones of Listeria monocytogenes
Authors: Lomonaco Sara , Knabel Stephen J. , Dalmasso Alessandra , Civera Tiziana , Bottero Maria Teresa
Published: July 2011

A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of
Listeria monocytogenes
and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of
L. monocytogenes
.
Persistent Phytoplankton Bloom in Lake St. Lucia (iSimangaliso Wetland Park, South Africa) Caused by a Cyanobacterium Closely Associated with the Genus Cyanothece (Synechococcaceae, Chroococcales)
Authors: Muir David G. , Perissinotto Renzo
Published: July 2011

Lake St. Lucia, iSimangaliso Wetland Park, South Africa, is the largest estuarine lake in Africa. Extensive use and manipulation of the rivers flowing into it have reduced freshwater inflow, and the lake has also been subject to a drought of 10 years. For much of this time, the estuary has been closed to the Indian Ocean, and salinities have progressively risen throughout the system, impacting the biotic components of the ecosystem, reducing zooplankton and macrobenthic biomass and diversity in particular. In June 2009, a bloom of a red/orange planktonic microorganism was noted throughout the upper reaches of Lake St. Lucia. The bloom persisted for at least 18 months, making it the longest such bloom on record. The causative organism was characterized by light and electron microscopy and by 16S rRNA sequencing and was shown to be a large, unicellular cyanobacterium most strongly associated with the genus
Cyanothece
. The extent and persistence of the bloom appears to be unique to Lake St. Lucia, and it is suggested that the organism's resistance to high temperatures, to intense insolation, and to hypersalinity as well as the absence of grazing pressure by salinity-sensitive zooplankton all contributed to its persistence as a bloom organism until a freshwater influx, due to exceptionally heavy summer rains in 2011, reduced the salinity for a sufficient length of time to produce a crash in the cyanobacterium population as a complex, low-salinity biota redeveloped.
Transcription Analysis of a Lantibiotic Gene Cluster from Bifidobacterium longum DJO10A
Authors: Lee Ju-Hoon , Li Xiulan , O'Sullivan Daniel J.
Published: July 2011

Bifidobacterium longumDJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of thelanAgene was undertaken. Comparative microarray analysis of broth and agar cultures ofB. longumDJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed thatlanAgene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures ofB. longumDJO10A inducedlanAgene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintainedlanAexpression. The expression oflanAwas log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis oflanArevealed a 284-bp 5′ untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp −75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of thislanAgene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.
Mechanism of Copper Surface Toxicity in Vancomycin-Resistant Enterococci following Wet or Dry Surface Contact
Authors: Warnes S. L. , Keevil C. W.
Published: July 2011

Contaminated touch surfaces have been implicated in the spread of hospital-acquired infections, and the use of biocidal surfaces could help to reduce this cross-contamination. In a previous study we reported the death of aqueous inocula of pathogenic
Enterococcus faecalis
or
Enterococcus faecium
isolates, simulating fomite surface contamination, in 1 h on copper alloys, compared to survival for months on stainless steel. In our current study we observed an even faster kill of over a 6-log reduction of viable enterococci in less than 10 min on copper alloys with a “dry” inoculum equivalent to touch contamination. We investigated the effect of copper(I) and copper(II) chelation and the quenching of reactive oxygen species on cell viability assessed by culture and their effects on genomic DNA, membrane potential, and respiration
in situ
on metal surfaces. We propose that copper surface toxicity for enterococci involves the direct or indirect action of released copper ionic species and the generation of superoxide, resulting in arrested respiration and DNA breakdown as the first stages of cell death. The generation of hydroxyl radicals by the Fenton reaction does not appear to be the dominant instrument of DNA damage. The bacterial membrane potential is unaffected in the early stages of wet and dry surface contact, suggesting that the membrane is not compromised until after cell death. These results also highlight the importance of correct surface cleaning protocols to perpetuate copper ion release and prevent the chelation of ions by contaminants, which could reduce the efficacy of the surface.
Investigation of the Electron Transport Chain to and the Catalytic Activity of the Diheme Cytochrome
c
Peroxidase CcpA of Shewanella oneidensis
Authors: Schütz Björn , Seidel Julian , Sturm Gunnar , Einsle Oliver , Gescher Johannes
Published: July 2011

Bacterial diheme
c
-type cytochrome peroxidases (BCCPs) catalyze the periplasmic reduction of hydrogen peroxide to water. The gammaproteobacterium
Shewanella oneidensis
produces the peroxidase CcpA under a number of anaerobic conditions, including dissimilatory iron-reducing conditions. We wanted to understand the function of this protein in the organism and its putative connection to the electron transport chain to ferric iron. CcpA was isolated and tested for peroxidase activity, and its structural conformation was analyzed by X-ray crystallography. CcpA exhibited
in vitro
peroxidase activity and had a structure typical of diheme peroxidases. It was produced in almost equal amounts under anaerobic and microaerophilic conditions. With 50 mM ferric citrate and 50 μM oxygen in the growth medium, CcpA expression results in a strong selective advantage for the cell, which was detected in competitive growth experiments with wild-type and Δ
ccpA
mutant cells that lack the entire
ccpA
gene due to a markerless deletion. We were unable to reduce CcpA directly with CymA, MtrA, or FccA, which are known key players in the chain of electron transport to ferric iron and fumarate but identified the small monoheme ScyA as a mediator of electron transport between CymA and BCCP. To our knowledge, this is the first detailed description of a complete chain of electron transport to a periplasmic
c
-type cytochrome peroxidase. This study furthermore reports the possibility of establishing a specific electron transport chain using
c
-type cytochromes.
Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD
+
-Auxotrophic Mutant
Authors: Zhou Yongjin , Wang Lei , Yang Fan , Lin Xinping , Zhang Sufang , Zhao Zongbao K.
Published: July 2011

NAD (NAD
+
) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD
+
level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in
Escherichia coli
cells that were genetically engineered to be NAD
+
auxotrophic. First, we expressed the
ntt4
gene encoding the NAD(H) transporter in the
E. coli
mutant YJE001, which had a deletion of the
nadC
gene responsible for NAD
+
de novo
biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD
+
. We then constructed the NAD
+
-auxotrophic mutant YJE003 by disrupting the essential gene
nadE
, which is responsible for the last step of NAD
+
biosynthesis in cells harboring the
ntt4
gene. The minimal NAD
+
level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD
+
, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD
+
was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD
+
concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.
Amoeba Host Model for Evaluation of Streptococcus suis Virulence
Authors: Bonifait Laetitia , Charette Steve J. , Filion Geneviève , Gottschalk Marcelo , Grenier Daniel
Published: July 2011

The Gram-positive bacterium
Streptococcus suis
is a major swine pathogen worldwide that causes meningitis, septicemia, and endocarditis. In this study, we demonstrate that the amoeba
Dictyostelium discoideum
can be a relevant alternative system to study the virulence of
S. suis
.
Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H
2
-Oxidizing Bacteria
Authors: Constant Philippe , Chowdhury Soumitra Paul , Hesse Laura , Pratscher Jennifer , Conrad Ralf
Published: July 2011

Streptomyces
soil isolates exhibiting the unique ability to oxidize atmospheric H
2
possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase
hhyS
and
hhyL
, respectively. Genome data mining revealed that the
hhyL
gene is unevenly distributed in the phyla
Actinobacteria
,
Proteobacteria
,
Chloroflexi
, and
Acidobacteria
. The
hhyL
gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H
2
-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H
2
oxidation activity and the
hhyL
gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10
6
to 10
8
hhyL
gene copies g (dry weight)
−1
. Assuming one
hhyL
gene copy per genome, the abundance of presumptive high-affinity H
2
-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H
2
oxidation activity measured in soil. Our data indicate that the abundance of the
hhyL
gene should not be taken as a reliable proxy for the uptake of atmospheric H
2
by soil, because high-affinity H
2
oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.
Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease
Authors: Galiana Eric , Marais Antoine , Mura Catherine , Industri Benoît , Arbiol Gilles , Ponchet Michel
Published: July 2011

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen
in vitro
and host disease. This approach was applied to a soilborne plant pathogen,
Phytophthora parasitica
, structured in a biofilm, for screening the microbial community from the rhizosphere of
Nicotiana tabacum
(the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A
Vorticella
species acted through a mutualistic interaction with
P. parasitica
, disseminating pathogenic material by leaving the biofilm. A
Phoma
species established an amensal interaction with
P. parasitica
, strongly suppressing disease by inhibiting
P. parasitica
germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.
Identification of a Toluene-Degrading Bacterium from a Soil Sample through H
2
18
O DNA Stable Isotope Probing
Authors: Woods Angela , Watwood Maribeth , Schwartz Egbert
Published: July 2011

DNA stable isotope probing (DNA-SIP) with H
2
18
O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H
2
18
O, H
2
16
O, H
2
16
O and 48 ppm toluene, or H
2
18
O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H
2
16
O. With extracts from soils to which only H
2
18
O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H
2
18
O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H
2
18
O into metabolic intermediates to form nucleic acids
de novo
. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader,
Rhodococcus jostii
RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be
Rhodococcus jostii
RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of
Rhodococcus jostii
RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H
2
18
O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.
Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei
Authors: Utter Christopher J. , Garcia Stacey A. , Milone Joseph , Bellofatto Vivian
Published: July 2011

Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5′ to 3′ exonuclease decay or 3′ to 5′ exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals,
Xenopus
oocytes, and higher plants. Trypanosomatids possess three different
PARN
genes,
PARN-1
, -
2
, and -
3
, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite
Trypanosoma brucei
. Here we show that
T. brucei
PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability
in vivo
, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.
YuaB Functions Synergistically with the Exopolysaccharide and TasA Amyloid Fibers To Allow Biofilm Formation by Bacillus subtilis
Authors: Ostrowski Adam , Mehert Angela , Prescott Alan , Kiley Taryn B. , Stanley-Wall Nicola R.
Published: July 2011

During biofilm formation by
Bacillus subtilis
, two extracellular matrix components are synthesized, namely, the TasA amyloid fibers and an exopolysaccharide. In addition, a small protein called YuaB has been shown to allow the biofilm to form. The regulatory protein DegU is known to initiate biofilm formation. In this report we show that the main role of DegU during biofilm formation is to indirectly drive the activation of transcription from the
yuaB
promoter. The N terminus of YuaB constitutes a signal peptide for the Sec transport system. Here we show that the presence of the signal peptide is required for YuaB function. In addition we demonstrate that upon export of YuaB from the cytoplasm, it localizes to the cell wall. We continue with evidence that increased production of TasA and the exopolysaccharide is not sufficient to overcome the effects of a mutation in
yuaB
, demonstrating the unique involvement of YuaB in forming a biofilm. In line with this, YuaB is not involved in correct synthesis, export, or polymerization of either the TasA amyloid fibers or the exopolysaccharide. Taken together, these findings identify YuaB as a protein that plays a novel role during biofilm formation. We hypothesize that YuaB functions synergistically with the known components of the biofilm matrix to facilitate the assembly of the biofilm matrix.
TrbB from Conjugative Plasmid F Is a Structurally Distinct Disulfide Isomerase That Requires DsbD for Redox State Maintenance
Authors: Hemmis Casey W. , Berkmen Mehmet , Eser Markus , Schildbach Joel F.
Published: July 2011

TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbBF plasmid inEscherichia colilacking DsbCE.coli, its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate thattrbBcan partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD fromE. colifor maintenance of its C-X-X-C redox active site motif.
Genome Sequences of Alicycliphilus denitrificans Strains BC and K601
T
Authors: Oosterkamp Margreet J. , Veuskens Teun , Plugge Caroline M. , Langenhoff Alette A. M. , Gerritse Jan , van Berkel Willem J. H. , Pieper Dietmar H. , Junca Howard , Goodwin Lynne A. , Daligault Hajnalka E. , Bruce David C. , Detter John C. , Tapia Roxanne , Han Cliff S. , Land Miriam L. , Hauser Loren J. , Smidt Hauke , Stams Alfons J. M.
Published: July 2011

Alicycliphilus denitrificans
strain BC and
A. denitrificans
strain K601
T
degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601
T
but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of
A. denitrificans
strains BC and K601
T
were sequenced. Here, we report the complete genome sequences of
A. denitrificans
strains BC and K601
T
.
RNase III-Dependent Expression of the
rpsO-pnp
Operon of Streptomyces coelicolor
Authors: Gatewood Marcha L. , Bralley Patricia , Jones George H.
Published: July 2011

We have examined the expression of the
rpsO-pnp
operon in an RNase III (
rnc
) mutant of
Streptomyces coelicolor
. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the
rnc
mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that
rpsO-pnp
mRNA levels increased in the
rnc
mutant by 1.6- to 4-fold compared with M145. This increase was observed in exponential, transition, and stationary phases, and the levels of the readthrough transcript, initiated upstream of
rpsO
in the
rpsO-pnp
operon; the
pnp
transcript, initiated in the
rpsO-pnp
intergenic region; and the
rpsO
transcript all increased. The increased levels of these transcripts in JSE1880 reflected increased chemical half-lives for each of the three. We demonstrated further that overexpression of the
rpsO
-
pnp
operon led to significantly higher levels of PNPase activity in JSE1880 compared to M145, reflecting the likelihood that PNPase expression is autoregulated in an RNase III-dependent manner in
S. coelicolor
. To explore further the increase in the level of the
pnp
transcript initiated in the intergenic region in JSE1880, we utilized that transcript as a substrate in assays employing purified
S. coelicolor
RNase III. These assays revealed the presence of hitherto-undiscovered sites of RNase III cleavage of the
pnp
transcript. The position of those sites was determined by primer extension, and they were shown to be situated in the loops of a stem-loop structure.
Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth
Authors: Nasrallah Gheyath K. , Riveroll Angela L. , Chong Audrey , Murray Lois E. , Lewis P. Jeffrey , Garduño Rafael A.
Published: July 2011

The Gram-negative intracellular pathogen
Legionella pneumophila
replicates in a membrane-bound compartment known as the
Legionella
-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with
L. pneumophila
expressing a translocation reporter consisting of the
Bordetella pertussis
adenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast
Saccharomyces cerevisiae
, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with
S
-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by
SPE2
) that is required for polyamine biosynthesis. Increasing the copy number of
SPE2
induced pseudohyphal growth in
S. cerevisiae
; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii)
L. pneumophila
may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced
L. pneumophila
replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote
L. pneumophila
proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in
L. pneumophila
, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of
L. pneumophila
.
Overexpression of LolCDE Allows Deletion of the Escherichia coli Gene Encoding Apolipoprotein
N
-Acyltransferase
Authors: Narita Shin-ichiro , Tokuda Hajime
Published: July 2011

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein
N
-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for
Escherichia coli
, a mutant lacking Lnt activity has not been isolated. However, we report here that
lnt
-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the
lnt
-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the
lnt
-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the
lnt
-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.
Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression
Authors: Brickman Timothy J. , Cummings Craig A. , Liew Sin-Yee , Relman David A. , Armstrong Sandra K.
Published: July 2011

Serological studies of patients with pertussis and the identification of antigenic
Bordetella pertussis
proteins support the hypothesis that
B. pertussis
perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these
B. pertussis
iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in
B. pertussis
gene transcript abundance in response to iron starvation
in vitro
. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system,
fbpABC
, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both
B. pertussis
strain Tohama I and
Bordetella bronchiseptica
strain RB50. In a survey of type III secretion system protein production by an assortment of
B. pertussis
laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only
Bordetella
iron transport gene expression but also the expression of other important virulence-associated genes.
KefF, the Regulatory Subunit of the Potassium Efflux System KefC, Shows Quinone Oxidoreductase Activity
Authors: Lyngberg Lisbeth , Healy Jessica , Bartlett Wendy , Miller Samantha , Conway Stuart J. , Booth Ian R. , Rasmussen Tim
Published: July 2011

Escherichia coli
and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g.,
N
-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.
Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. pomaceae Lectotype Strain ATCC 29192
Authors: Kouvelis Vassili N. , Davenport Karen W. , Brettin Thomas S. , Bruce David , Detter Chris , Han Cliff S. , Nolan Matt , Tapia Roxanne , Damoulaki Agni , Kyrpides Nikos C. , Typas Milton A. , Pappas Katherine M.
Published: July 2011

Zymomonas mobilis
is an alphaproteobacterium studied for bioethanol production. Different strains of this organism have been hitherto sequenced; they all belong to the
Z. mobilis
subsp.
mobilis
taxon. Here we report the finished and annotated genome sequence of strain ATCC 29192, a cider-spoiling agent isolated in the United Kingdom. ATCC 29192 is the lectotype of the second-best-characterized subspecies of
Z. mobilis
,
Z. mobilis
subsp.
pomaceae
. The nucleotide sequence of ATCC 29192 deviates from that of
Z. mobilis
subsp.
mobilis
representatives, which justifies its distinct taxonomic positioning and proves particularly useful for comparative and functional genomic analyses.
Complete Genome Sequence of the Commensal Streptococcus salivarius Strain JIM8777
Authors: Guédon Eric , Delorme Christine , Pons Nicolas , Cruaud Corinne , Loux Valentin , Couloux Arnaud , Gautier Céline , Sanchez Nicolas , Layec Séverine , Galleron Nathalie , Almeida Mathieu , van de Guchte Maarten , Kennedy Sean P. , Ehrlich S. Dusko , Gibrat Jean-François , Wincker Patrick , Renault Pierre
Published: July 2011

The commensal bacterium
Streptococcus salivarius
is a prevalent species of the human oropharyngeal tract with an important role in oral ecology. Here, we report the complete 2.2-Mb genome sequence and annotation of strain JIM8777, which was recently isolated from the oral cavity of a healthy, dentate infant.
Complete Genome Sequence of the Clinical Streptococcus salivarius Strain CCHSS3
Authors: Delorme Christine , Guédon Eric , Pons Nicolas , Cruaud Corinne , Couloux Arnaud , Loux Valentin , Chiapello Hélène , Poyart Claire , Gautier Céline , Sanchez Nicolas , Almeida Mathieu , Kennedy Sean P. , Ehrlich S. Dusko , Gibrat Jean-François , Wincker Patrick , Renault Pierre
Published: July 2011

Streptococcus salivarius
is a commensal species commonly found in the human oral cavity and digestive tract, although it is also associated with human infections such as meningitis, endocarditis, and bacteremia. Here, we report the complete sequence of
S. salivarius
strain CCHSS3, isolated from human blood.
Draft Genome Sequence of the Paenibacillus polymyxa Type Strain (ATCC 842
T
), a Plant Growth-Promoting Bacterium
Authors: Jeong Haeyoung , Park Soo-Young , Chung Won-Hyong , Kim Sun Hong , Kim Namshin , Park Seung-Hwan , Kim Jihyun F.
Published: July 2011

Paenibacillus polymyxa
is an endospore-forming Gram-positive soil bacterium that is well-known for its ability to promote plant growth. Here we report the draft genome sequence of
P. polymyxa
ATCC 842
T
, the type strain of the species
P. polymyxa
, and the family
Paenibacillaceae
. The
P. polymyxa
genome contains a repertoire of biosynthetic genes for antibiotics and hydrolytic enzymes that account for its beneficial effects in the rhizosphere to the host plants it associates with.
Genome Sequence of the Nonpathogenic Listeria monocytogenes Serovar 4a Strain M7
Authors: Chen Jianshun , Xia Ye , Cheng Changyong , Fang Chun , Shan Ying , Jin Gulei , Fang Weihuan
Published: July 2011

This report presents the complete and annotated genome sequence of the naturally nonpathogenicListeria monocytogenesserovar 4a strain M7, isolated from cow's milk in Zhejiang province, China.
Draft Genome Sequence of Gordonia neofelifaecis NRRL B-59395, a Cholesterol-Degrading Actinomycete
Authors: Ge Fanglan , Li Wei , Chen Guiying , Liu Yuchang , Zhang Guangxiang , Yong Bin , Wang Qiong , Wang Nan , Huang Zhumei , Li Weitian , Wang Jing , Wu Cheng , Xie Qian , Liu Gang
Published: July 2011

We report a draft sequence of the genome of
Gordonia neofelifaecis
NRRL B-59395, a cholesterol-degrading actinomycete isolated from fresh feces of a clouded leopard (
Neofelis nebulosa
). As predicted, the reported genome contains several gene clusters for cholesterol degradation. This is the second available genome sequence of the family
Gordoniaceae
.
Genome Sequence of Lactobacillus salivarius GJ-24, a Probiotic Strain Isolated from Healthy Adult Intestine
Authors: Cho Yong-Joon , Choi Jae Kyoung , Kim Ji-Hee , Lim Yea-Seul , Ham Jun-Sang , Kang Dae-Kyung , Chun Jongsik , Paik Hyun-Dong , Kim Geun-Bae
Published: July 2011

The draft genome sequence of
Lactobacillus salivarius
GJ-24 isolated from the feces of healthy adults was determined. Its properties, including milk fermentation activity and bacteriocin production, suggest its potential uses as a probiotic lactic acid bacterium and start culture for dairy products.