bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Throughput and Quantitative Procedure for Determining Sources of
Escherichia coli
in Waterways by Using Host-Specific DNA Marker Genes
Authors: Yan Tao , Hamilton Matthew J. , Sadowsky Michael J.
Published: December 2006

Escherichia coli
is currently used as an indicator of fecal pollution and to assess water quality. While several genotypic techniques have been used to determine potential sources of fecal bacteria impacting waterways and beaches, they do not allow for the rapid analysis of a large number of samples in a relatively short period of time. Here we report that gene probes identified by Hamilton and colleagues (M. J. Hamilton, T. Yan, and M. J. Sadowsky, Appl. Environ. Microbiol.
72:
4012-4019, 2006) were useful for the development of a high-throughput and quantitative macroarray hybridization system to determine numbers of
E. coli
bacteria originating from geese/ducks. The procedure we developed, using a QBot robot for picking and arraying of colonies, allowed us to simultaneously analyze up to 20,736
E. coli
colonies from water samples, with minimal time and human input. Statistically significant results were obtained by analyzing 700
E. coli
colonies per water sample, allowing for the analysis of approximately 30 sites per macroarray. Macroarray hybridization studies done on
E. coli
collected from water samples obtained from two urban Minnesota lakes and one rural South Carolina lake indicated that geese/ducks contributed up to 51% of the fecal bacteria in the urban lake water samples, and the level was below the detection limit in the rural lake water sample. This technique, coupled with the use of other host source-specific gene probes, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of
E. coli
in waterways and on beaches.
Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids
Authors: Sousa Diana Z. , Pereira M. Alcina , Stams Alfons J. M. , Alves M. Madalena , Smidt Hauke
Published: December 2006

Anaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria within
Syntrophomonadaceae
and
Syntrophobacteraceae
families. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with the
Syntrophomonas
genus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.
The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation
Authors: Lauro Federico M. , Chastain Roger A. , Blankenship Lesley E. , Yayanos A. Aristides , Bartlett Douglas H.
Published: December 2006

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few γ-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.
CO
2
Uptake and Fixation by Endosymbiotic Chemoautotrophs from the Bivalve
Solemya velum
Authors: Scott Kathleen M. , Cavanaugh Colleen M.
Published: December 2006

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve
Solemya velum
. Symbiont-enriched suspensions were generated by homogenization of
S. velum
gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with
14
C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO
2
and not with HCO
3
−
concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO
2
was 28 ± 3 μM, and the average maximal velocity was 50.8 ± 7.1 μmol min
−1
g of protein
−1
. The reliance of
S. velum
symbionts on extracellular CO
2
is consistent with their intracellular lifestyle, since HCO
3
−
utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO
2
may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.
Species-Specific Secondary Metabolite Production in Marine Actinomycetes of the Genus
Salinispora
Authors: Jensen Paul R. , Williams Philip G. , Oh Dong-Chan , Zeigler Lisa , Fenical William
Published: December 2006

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus
Salinispora
. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.
Metaproteomics Approach To Study the Functionality of the Microbiota in the Human Infant Gastrointestinal Tract
Authors: Klaassens Eline S. , de Vos Willem M. , Vaughan Elaine E.
Published: December 2006

A metaproteomics approach comprising two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) was applied to the largely uncultured infant fecal microbiota for the first time. The fecal microbial metaproteome profiles changed over time, and one protein spot contained a peptide sequence that showed high similarity to those of bifidobacterial transaldolases.
Polyphosphate Accumulation by
Pseudomonas putida
CA-3 and Other Medium-Chain-Length Polyhydroxyalkanoate-Accumulating Bacteria under Aerobic Growth Conditions
Authors: Tobin Karen M. , McGrath John W. , Mullan Alan , Quinn John P. , O'Connor Kevin E.
Published: December 2006

Pseudomonas putida
CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating
Pseudomonas
strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these
Pseudomonas
strains.
Expression of a Novel Small Antimicrobial Protein from the Seeds of Motherwort (
Leonurus japonicus
) Confers Disease Resistance in Tobacco
Authors: Yang Xingyong , Xiao Yuehua , Wang Xiaowen , Pei Yan
Published: December 2006

Medicinal
plants are valuable resources of natural antimicrobial materials. A
novel small protein with antimicrobial activities, designated LJAMP1,
was purified from the seeds of a medicinal herb, motherwort
(
Leonurus japonicus
Houtt). LJAMP1 is a heat-stable protein
with a molecular mass of 7.8 kDa and a determined isoelectric point of
8.2. In vitro assays showed that LJAMP1 inhibits the growth of an array
of fungi and bacteria. The hyphal growth inhibition by LJAMP1 was more
evident against hyphomycete fungi, such as
Alternaria
alternata
,
Cercospora personata
, and
Aspergillus
niger
. The N-terminal amino acid sequence of LJAMP1 was
determined, and its coding gene was consequently cloned by the rapid
amplification of cDNA ends. The gene
LJAMP1
has no intron and
encodes a polypeptide of 95 amino acids, in which the first 27 residues
was deduced as a signal peptide. The mature LJAMP1 shows relatively low
identity to plant napin-like storage proteins. Northern blot assays
revealed that
LJAMP1
is expressed preferentially in seeds.
Bioassays in transgenic tobacco demonstrated that that overexpression
of
LJAMP1
significantly enhanced the resistance of tobacco
against not only the fungal pathogen
A. alternata
but also the
bacterial pathogen
Ralstonia solanacearum
, while no visible
alteration in plant growth and development was
observed.
Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions
Authors: Blieck Lies , Toye Geert , Dumortier Françoise , Verstrepen Kevin J. , Delvaux Freddy R. , Thevelein Johan M. , Van Dijck Patrick
Published: December 2006

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (
Saccharomyces pastorianus
) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.
Coral Disease Diagnostics: What's between a Plague and a Band?
Authors: Ainsworth T. D. , Kramasky-Winter E. , Loya Y. , Hoegh-Guldberg O. , Fine M.
Published: December 2006

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.
Cyclohexane Carboxylate and Benzoate Formation from Crotonate in
Syntrophus aciditrophicus
Authors: Mouttaki Housna , Nanny Mark A. , McInerney Michael J.
Published: December 2006

The anaerobic, syntrophic bacterium
Syntrophus aciditrophicus
grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-
13
C]crotonate was metabolized to [1,2-
13
C]acetate and [1,2,3,4,5,7-
13
C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [
13
C]sodium bicarbonate formed [6-
13
C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-
13
C]crotonate, the
m/z
-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the
m/z
-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [
13
C]sodium bicarbonate and unlabeled crotonate, the
m/z
-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-
13
C]acetate and unlabeled crotonate, the
m/z
-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the
m/z
-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO
2
addition, rather than the use of the intact four-carbon skeleton of crotonate.
Benzoate Catabolite Repression of the Phthalate Degradation Pathway in
Rhodococcus
sp. Strain DK17
Authors: Choi Ki Young , Zylstra Gerben J. , Kim Eungbin
Published: December 2006

Rhodococcus
sp. strain DK17 exhibits a catabolite repression-like response when provided simultaneously with benzoate and phthalate as carbon and energy sources. Benzoate in the medium is depleted to detection limits before the utilization of phthalate begins. The transcription of the genes encoding benzoate and phthalate dioxygenase paralleled the substrate utilization profile. Two mutant strains with defective benzoate dioxygenases were unable to utilize phthalate in the presence of benzoate, although they grew normally on phthalate in the absence of benzoate.
Chaperone Hsp31 Contributes to Acid Resistance in Stationary-PhaseEscherichia coli
Authors: Mujacic Mirna , Baneyx François
Published: December 2006

Hsp31, the product of the σS- and σD-dependenthchAgene, is a heat-inducible chaperone implicated in the management of protein misfolding at high temperatures. We show here that Hsp31 plays an important role in the acid resistance of starvedEscherichia colibut that it has little influence on oxidative-stress survival.
Homologous Subunits of 1,3-Beta-Glucan Synthase Are Important for Spore Wall Assembly in
Saccharomyces cerevisiae
Authors: Ishihara Satoru , Hirata Aiko , Nogami Satoru , Beauvais Anne , Latge Jean-Paul , Ohya Yoshikazu
Published: December 2006

During
sporulation in
Saccharomyces cerevisiae
, the four
haploid nuclei are encapsulated within multilayered spore walls.
Glucan, the major constituent of the spore wall, is synthesized by
1,3-β-glucan synthase, which is composed of a putative
catalytic subunit encoded by
FKS1
and
FKS2
. Although
another homolog, encoded by
FKS3
, was identified by homology
searching, its function is unknown. In this report, we show that
FKS2
and
FKS3
are required for spore wall assembly.
The ascospores of
fks2
and
fks3
mutants were
enveloped by an abnormal spore wall with reduced resistance to diethyl
ether, elevated temperatures, and ethanol. However, deletion of the
FKS1
gene did not result in a defective spore wall. The
construction of fusion genes that expressed Fks1p and Fks2p under the
control of the
FKS2
promoter revealed that asci transformed
with
FKS2p
-driven Fks1p and Fks2p were resistant to elevated
temperatures, which suggests that the expression of
FKS2
plays
an important role in spore wall assembly. The expression of
FKS1p
-driven Fks3p during vegetative growth did not affect
1,3-β-glucan synthase activity in vitro but effectively
suppressed the growth defect of the temperature-sensitive
fks1
mutant by stabilizing Rho1p, which is a regulatory subunit of glucan
synthase. Based on these results, we propose that
FKS2
encodes
the primary 1,3-β-glucan synthase in sporulation and that
FKS3
is required for normal spore wall formation because it
affects the upstream regulation of 1,3-β-glucan
synthase.
Small-Subunit rRNA Processome Proteins Are Translationally Regulated during Differentiation of
Trypanosoma cruzi
Authors: Nardelli Sheila Cristina , Ávila Andréa Rodrigues , Freund Aline , Motta Maria Cristina , Manhães Lauro , de Jesus Teresa Cristina Leandro , Schenkman Sergio , Fragoso Stenio Perdigão , Krieger Marco Aurélio , Goldenberg Samuel , Dallagiovanna Bruno
Published: December 2006

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite
Trypanosoma cruzi
. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding
T. cruzi
protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in
T. cruzi
also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.
Two-Component Response Regulators Ssk1p and Skn7p Additively Regulate High-Osmolarity Adaptation and Fungicide Sensitivity in
Cochliobolus heterostrophus
Authors: Izumitsu Kosuke , Yoshimi Akira , Tanaka Chihiro
Published: December 2006

Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungus unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes Ch
Ssk1
and Ch
Skn7
in the southern corn leaf blight fungus
Cochliobolus heterostrophus
. Both Ch
Ssk1
- and Ch
Skn7
-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The Ch
ssk1
Ch
skn7
double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1- and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from that of yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.
The Type IV Secretion System ofSinorhizobium melilotiStrain 1021 Is Required for Conjugation but Not for Intracellular Symbiosis
Authors: Jones Kathryn M. , Lloret Javier , Daniele Joseph R. , Walker Graham C.
Published: December 2006

The type IV secretion system (T4SS) of the plant intracellular symbiontSinorhizobium meliloti1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.
Genomic Plasticity of therrn-nqrFIntergenic Segment in theChlamydiaceae
Authors: Liu Zhi , Rank Roger , Kaltenboeck Bernhard , Magnino Simone , Dean Deborah , Burall Laurel , Plaut Roger D. , Read Timothy D. , Myers Garry , Bavoil Patrik M.
Published: December 2006

InChlamydiaceae, the nucleotide sequence between the 5S rRNA gene and the gene for subunit F of the Na+-translocating NADH-quinone reductase (nqrFordmpP) has varied lengths and gene contents. We analyzed this site in 45Chlamydiaceaestrains having diverse geographical and pathological origins and including members of all nine species.
Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System
Authors: Francetic Olivera , Buddelmeijer Nienke , Lewenza Shawn , Kumamoto Carol A. , Pugsley Anthony P.
Published: December 2006

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from
Klebsiella oxytoca
. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in
Escherichia coli
derivatives defective in different protein targeting and export factors. The β-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the
dsbA
,
dsbB
, or all
sec
genes tested except
secB
were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in
secA
,
secY
,
secE
, and
secD
genes, including some novel alleles of
secY
and
secD
. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.
Hfq Modulates the σ
E
-Mediated Envelope Stress Response and the σ
32
-Mediated Cytoplasmic Stress Response in
Escherichia coli
Authors: Guisbert Eric , Rhodius Virgil A. , Ahuja Nidhi , Witkin Emily , Gross Carol A.
Published: December 2006

Hfq, a chaperone for small noncoding RNAs, regulates many processes in
Escherichia coli
, including the σ
S
-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the σ
E
- and σ
32
-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the σ
E
-mediated envelope stress response and are defective in σ
E
-mediated repression of outer membrane proteins. We also show that the σ
32
-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the “long-term adaptation” of σ
32
to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in
E. coli
.
Role of a Conserved Membrane Glycine Residue in a Dicarboxylate Transporter from
Sinorhizobium meliloti
Authors: Trainer Maria A. , Yurgel Svetlana N. , Kahn Michael L.
Published: December 2006

Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.
Whole-Genome Analysis of the Methyl
tert
-Butyl Ether-Degrading Beta-Proteobacterium
Methylibium petroleiphilum
PM1
Authors: Kane Staci R. , Chakicherla Anu Y. , Chain Patrick S. G. , Schmidt Radomir , Shin Maria W. , Legler Tina C. , Scow Kate M. , Larimer Frank W. , Lucas Susan M. , Richardson Paul M. , Hristova Krassimira R.
Published: December 2006

Methylibium petroleiphilum
PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl
tert
-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C
5
to C
12
) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an ∼4-Mb circular chromosome and an ∼600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G+C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (∼99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in
M. petroleiphilum
and other MTBE-degrading beta-proteobacteria.
A Protein from a Parasitic Microorganism,
Rickettsia prowazekii
, Can Cleave the Signal Sequences of Proteins Targeting Mitochondria
Authors: Kitada Sakae , Uchiyama Tsuneo , Funatsu Tomoyuki , Kitada Yumiko , Ogishima Tadashi , Ito Akio
Published: December 2006

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the α and β subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with β-MPP but barely contained any of the glycine-rich loop region characteristic of α-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast β-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.
Possible Nonconductive Role of
Geobacter sulfurreducens
Pilus Nanowires in Biofilm Formation
Authors: Reguera Gemma , Pollina Rachael B. , Nicoll Julie S. , Lovley Derek R.
Published: December 2006

Geobacter sulfurreducens
required expression of electrically conductive pili to form biofilms on Fe(III) oxide surfaces, but pili were also essential for biofilm development on plain glass when fumarate was the sole electron acceptor. Furthermore, pili were needed for cell aggregation in agglutination studies. These results suggest that the pili of
G. sulfurreducens
also have a structural role in biofilm formation.
Identification of Mycobacterial σ Factor Binding Sites by Chromatin Immunoprecipitation Assays
Authors: Rodrigue Sébastien , Brodeur Joëlle , Jacques Pierre-Étienne , Gervais Alain L. , Brzezinski Ryszard , Gaudreau Luc
Published: December 2006

Mycobacterium tuberculosis
and
Mycobacterium bovis
are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial σ factors. Chromatin immunoprecipitation assays were performed with
M. bovis
BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the
M. tuberculosis
H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for σ
F
, σ
C
, and σ
K
, and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of σ factor genes. Our results revealed that many σ factors are expressed from autoregulated promoters.
Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole
Authors: Yao Shiyin , Helinski Donald R. , Toukdarian Aresa
Published: December 2006

The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of
Escherichia coli
. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (
sopABC
) or RK2 (O
B1
incC korB
) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of
par
regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.
Transcription from the P3Promoter of theBacillus subtilis spxGene Is Induced in Response to Disulfide Stress
Authors: Leelakriangsak Montira , Zuber Peter
Published: December 2006

Thespxgene ofBacillus subtilisencodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with theyjbCgene. Thespxgene DNA complements anspxnull mutation with respect to disulfide stress resistance, suggesting thatspxis transcribed from a promoter located in the intergenic region ofyjbCandspx. Transcription of theyjbC-spxoperon has been reported to be driven by four promoters, three (P1, P2, and PB) residing upstream ofyjbCand one (PM) located in the intergenic region betweenyjbCandspx. Primer extension analysis uncovered a second intergenic promoter, P3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P3is utilized by the σAform of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P3did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position −40 of the P3promoter start site. Deletion mutants with endpoints 3′ to the P3transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P3promoter sequence. Point mutations characterized bybgaBfusion expression and primer extension analyses uncovered evidence for a secondcis-acting site in the P3promoter sequence itself. The data indicate thatspxtranscription is under negative transcriptional control that is reversed when disulfide stress is encountered.
Dual Negative Control ofspxTranscription Initiation from the P3Promoter by Repressors PerR and YodB inBacillus subtilis
Authors: Leelakriangsak Montira , Kobayashi Kazuo , Zuber Peter
Published: December 2006

Thespxgene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress inBacillus subtilis. It resides in theyjbC-spxoperon and is transcribed from at least five promoters located in theyjbCregulatory region or in theyjbC-spxintergenic region. Induction ofspxtranscription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P3promoter located upstream of thespxcoding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target thecis-acting negative control elements of the P3promoter. Expression of anspx-bgaBfusion carrying the P3promoter is elevated in ayodBorperRmutant, and an additive increase in expression was observed in ayodB perRdouble mutant. Primer extension analysis ofspxRNA shows the same additive increase in P3transcript levels inyodB perRmutant cells. Purified YodB and PerR repressspxtranscription in vitro when wild-typespxP3promoter DNA was used as a template. Point mutations at positions within the P3promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P3−10 and −35 regions, while PerR binds to a region downstream of the P3transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.
Only One of Five
groEL
Genes Is Required for Viability and Successful Symbiosis in
Sinorhizobium meliloti
Authors: Bittner Alycia N. , Foltz Amanda , Oke Valerie
Published: December 2006

Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its cochaperone, GroES, including all of the fully sequenced root-nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in
Sinorhizobium meliloti
there are four
groESL
operons and one
groEL
gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of
groEL
mutations. Although a double
groEL1 groEL2
mutant cannot be constructed, we demonstrate that the quadruple
groEL1 groESL3 groEL4 groESL5
and
groEL2 groESL3 groEL4 groESL5
mutants are viable. Therefore, like
E. coli
and other species,
S. meliloti
requires only one
groEL
gene for viability, and either
groEL1
or
groEL2
will suffice. The
groEL1 groESL5
double mutant is more severely affected for growth at both 30°C and 40°C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple
groEL2 groESL3 groEL4 groESL5
mutant acts like the wild type, but the quadruple
groEL1 groESL3 groEL4 groESL5
mutant acts like the
groEL1
single mutant, which cannot fully induce
nod
gene expression and forms ineffective nodules. Therefore, the only
groEL
gene required for symbiosis is
groEL1
. However, we show that the other
groE
genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that
groESL1
encodes the housekeeping GroEL/GroES chaperone and that
groESL5
is specialized for stress response.
Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of
Bacillus subtilis
Spores Triggered by Dodecylamine or Lysozyme
Authors: Vepachedu Venkata Ramana , Setlow Peter
Published: December 2006

The release of dipicolinic acid (DPA) during the germination of
Bacillus subtilis
spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of
B. subtilis
spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl
2
. Spores carrying temperature-sensitive mutants in the
spoVA
operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca
2+
almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca
2+
and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca
2+
-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca
2+
-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.
Identification of Genes Involved in Swarming Motility Using aPseudomonas aeruginosaPAO1 Mini-Tn5-luxMutant Library
Authors: Overhage Joerg , Lewenza Shawn , Marr Alexandra K. , Hancock Robert E. W.
Published: December 2006

During a screening of a mini-Tn5-luxCDABEtransposon mutant library ofPseudomonas aeruginosaPAO1 for alterations in swarming motility, 36 mutants were identified with Tn5insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes inP. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.
Evidence that RpoS (σ
S
) in
Borrelia burgdorferi
Is Controlled Directly by RpoN (σ
54
/σ
N
)
Authors: Smith Alexandra H. , Blevins Jon S. , Bachlani Gulnaz N. , Yang Xiaofeng F. , Norgard Michael V.
Published: December 2006

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in
Borrelia burgdorferi
. However, evidence to support whether RpoN controls
rpoS
directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls
rpoS
in
B. burgdorferi
.
Biochemical and Genetic Analysis of the γ-Resorcylate (2,6-Dihydroxybenzoate) Catabolic Pathway in
Rhizobium
sp. Strain MTP-10005: Identification and Functional Analysis of Its Gene Cluster
Authors: Yoshida Masahiro , Oikawa Tadao , Obata Hitoshi , Abe Katsumasa , Mihara Hisaaki , Esaki Nobuyoshi
Published: December 2006

We identified a gene cluster that is involved in the γ-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium
Rhizobium
sp. strain MTP-10005. The cluster consists of the
graRDAFCBEK
genes, and
graA
,
graB
,
graC
, and
graD
were heterologously expressed in
Escherichia coli
. Enzymological studies showed that
graD
,
graA
,
graC
, and
graB
encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that
graE
,
graR
, and
graK
encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of
graF
, which encodes γ-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 ± 0.82] × 10
−4
) of
graF
was detected in the total RNA of the cells after one hour of cultivation when γ-resorcylate was used as the sole carbon source. Reverse transcription-PCR of
graDAFCBE
showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by γ-resorcylate. These results suggested that the
graDAFCBE
genes are responsible as an operon for the growth of
Rhizobium
sp. strain MTP-10005 on γ-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the γ-resorcylate catabolic pathway in an aerobic bacterium.