bluuebunny/crossref_metadata_2025 huggingface dataset:
[FeFe] Hydrogenase Genetic Diversity Provides Insight into Molecular Adaptation in a Saline Microbial Mat Community
Authors: Boyd Eric S. , Spear John R. , Peters John W.
Published: May 2009

Degenerate primers for the [FeFe] hydrogenase (
hydA
) were developed and used in PCRs to examine
hydA
in microbial mats that inhabit saltern evaporative ponds in Guerrero Negro (GN), Mexico. A diversity of deduced HydA was discovered that revealed unique variants, which may reflect adaptation to the environmental conditions present in GN.
Importance of
trmE
for Growth of the Psychrophile
Pseudomonas syringae
at Low Temperatures
Authors: Singh Ashish K. , Pindi Pavan Kumar , Dube Smita , Sundareswaran V. R. , Shivaji S.
Published: May 2009

Transposon mutagenesis of
Pseudomonas syringae
Lz4W, a psychrophilic bacterium capable of growing at temperatures between 2 and 30°C, yielded 30 cold-sensitive mutants, and CSM1, one of these cold-sensitive mutants, was characterized. Growth of CSM1 was retarded when it was cultured at 4°C but not when it was cultured at 22°C and 28°C compared to the growth of wild-type cells, indicating that CSM1 is a cold-sensitive mutant of
P. syringae
Lz4W. The mutated gene in CSM1 was identified as
trmE
(coding for tRNA modification GTPase), and evidence is provided that this gene is induced at low temperatures. Further, the cold-inducible nature of the
trmE
promoter was demonstrated. In addition, the transcription start site and the various regulatory elements of the
trmE
promoter, such as the −10 region, −35 region, UP element, cold box, and DEAD box, were identified, and the importance of these regulatory elements in promoter activity were confirmed. The importance of
trmE
in rapid adaptation to growth at low temperatures was further highlighted by plasmid-mediated complementation that alleviated the cold-sensitive phenotype of CSM1.
Aerobic Mineralization of Hexachlorobenzene by Newly Isolated Pentachloronitrobenzene-Degrading
Nocardioides
sp. Strain PD653
Authors: Takagi Kazuhiro , Iwasaki Akio , Kamei Ichiro , Satsuma Koji , Yoshioka Yuichi , Harada Naoki
Published: May 2009

A novel aerobic pentachloronitrobenzene-degrading bacterium,
Nocardioides
sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of
14
CO
2
from [U-ring-
14
C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.
Staphylococcus aureus
Virulence Expression Is Impaired by
Lactococcus lactis
in Mixed Cultures
Authors: Even Sergine , Charlier Cathy , Nouaille Sébastien , Ben Zakour Nouri L. , Cretenet Marina , Cousin Fabien J. , Gautier Michel , Cocaign-Bousquet Muriel , Loubière Pascal , Le Loir Yves
Published: May 2009

Staphylococcus aureus
is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as
Lactococcus lactis
, a lactic acid bacterium widely used in the dairy industry, can modulate
S. aureus
proliferation and virulence expression. We developed a dedicated
S. aureus
microarray to investigate the effect of
L. lactis
on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of
S. aureus
in mixed cultures with
L. lactis
in a chemically defined medium held at a constant pH (6.6). Under these conditions,
L. lactis
hardly affected
S. aureus
growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the
agr
locus,
sarA
, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of
L. lactis
antagonistic potential for
S. aureus
and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.
Fermentation pH Influences the Physiological-State Dynamics of
Lactobacillus bulgaricus
CFL1 during pH-Controlled Culture
Authors: Rault Aline , Bouix Marielle , Béal Catherine
Published: May 2009

This study aims at better understanding the effects of fermentation pH and harvesting time on
Lactobacillus bulgaricus
CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.
CO2Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring
Authors: Boyd Eric S. , Leavitt William D. , Geesey Gill G.
Published: May 2009

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (Sofloc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO2uptake of 21.3 ± 11.9 μg of C 107cells−1h−1. When extrapolated over the estimated total quantity of Sofloc at the spring's source, the Sofloc-associated microbial community accounted for the uptake of 121 mg of C h−1at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated bySynechococcusspp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO2by the Sofloc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the Sofloc-associated community were related to chemolithoautotrophicHydrogenobaculumstrains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO2in this geothermal habitat.
Bar-Coded Pyrosequencing Reveals Shared Bacterial Community Properties along the Temperature Gradients of Two Alkaline Hot Springs in Yellowstone National Park
Authors: Miller Scott R. , Strong Aaron L. , Jones Kenneth L. , Ungerer Mark C.
Published: May 2009

An understanding of how communities are organized is a fundamental goal of ecology but one which has historically been elusive for microbial systems. We used a bar-coded pyrosequencing approach targeting the V3 region of the bacterial small-subunit rRNA gene to address the factors that structure communities along the thermal gradients of two alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park. The filtered data set included a total of nearly 34,000 sequences from 39 environmental samples. Each was assigned to one of 391 operational taxonomic units (OTUs) identified by their unique V3 sequence signatures. Although the two hot springs differed in their OTU compositions, community resemblance and diversity changed with strikingly similar dynamics along the two outflow channels. Two lines of evidence suggest that these community properties are controlled primarily by environmental temperature. First, community resemblance decayed exponentially with increasing differences in temperature between samples but was only weakly correlated with physical distance. Second, diversity decreased with increasing temperature at the same rate along both gradients but was uncorrelated with other measured environmental variables. This study also provides novel insights into the nature of the ecological interactions among important taxa in these communities. A strong negative association was observed between cyanobacteria and the
Chloroflexi
, which together accounted for ∼70% of the sequences sampled. This pattern contradicts the longstanding hypothesis that coadapted lineages of these bacteria maintain tightly cooccurring distributions along these gradients as a result of a producer-consumer relationship. We propose that they instead compete for some limiting resource(s).
Direct Effects of UV-B Radiation on the Freshwater Heterotrophic NanoflagellateParaphysomonassp
Authors: Macaluso Amy L. , Mitchell David L. , Sanders Robert W.
Published: May 2009

The formation of DNA photoproducts in organisms exposed to ambient levels of UV-B radiation can lead to death and/or reduced population growth in aquatic systems. Dependence on photoenzymatic repair to reverse DNA damage caused by UV-B radiation is demonstrated forParaphysomonassp., a member of a widely distributed genus of heterotrophic nanoflagellates. At 20°C,Paraphysomonassp. was exposed to a range of UV-B intensities encountered in natural systems. Populations of the flagellate survived and grew in a dose-dependent manner, but only when simultaneously exposed to photorepair radiation (PRR). In contrast, flagellates exposed to UV-B at 15°C suffered 100% mortality except at the lowest UV-B level (with PRR) tested, which suggested a photorepair temperature optimum above 15°C. After acute UV-B exposures, DNA damage (measured as the formation of pyrimidine dimers) was reduced only in organisms that underwent subsequent exposure to PRR. Populations kept in the dark after UV-B exposure maintained the initial levels of pyrimidine dimers. These results are the first to demonstrate the reliance of a heterotrophic flagellate on photoenzymatic DNA repair for survival from UV-B exposure.
Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing “
Leptospirillum rubarum
” (Group II) and “
Leptospirillum ferrodiazotrophum
” (Group III) Bacteria in Acid Mine Drainage Biofilms
Authors: Goltsman Daniela S. Aliaga , Denef Vincent J. , Singer Steven W. , VerBerkmoes Nathan C. , Lefsrud Mark , Mueller Ryan S. , Dick Gregory J. , Sun Christine L. , Wheeler Korin E. , Zemla Adam , Baker Brett J. , Hauser Loren , Land Miriam , Shah Manesh B. , Thelen Michael P. , Hettich Robert L. , Banfield Jillian F.
Published: May 2009

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing
Leptospirillum
group II and III bacteria (phylum
Nitrospirae
) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of
Leptospirillum
groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both
Leptospirillum
types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in
Leptospirillum
group III, consistent with its distribution in gradients within biofilms. Interestingly,
Leptospirillum
group II uses a methyl-dependent and
Leptospirillum
group III a methyl-independent response pathway. Although only
Leptospirillum
group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of
Leptospirillum
groups II and III.
Analysis of Flagellar Phosphoproteins from
Chlamydomonas reinhardtii
Authors: Boesger Jens , Wagner Volker , Weisheit Wolfram , Mittag Maria
Published: May 2009

Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga
Chlamydomonas reinhardtii
has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.
Spliceosomal Proteomics in
Trypanosoma brucei
Reveal New RNA Splicing Factors
Authors: Luz Ambrósio Daniela , Lee Ju Huck , Panigrahi Aswini K. , Nguyen Tu Ngoc , Cicarelli Regina Maria Barretto , Günzl Arthur
Published: May 2009

In trypanosomatid parasites, spliced leader (SL)
trans
splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are
trans
spliced, intron removal by
cis
splicing is extremely rare and predicted to occur in only four pre-mRNAs.
trans
- and
cis
-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of
trans
splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in
Trypanosoma brucei
and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.
Chitin in Diatoms and Its Association with the Cell Wall
Authors: Durkin Colleen A. , Mock Thomas , Armbrust E. Virginia
Published: May 2009

Chitin is a globally abundant polymer widely distributed throughout eukaryotes that has been well characterized in only a few lineages. Diatoms are members of the eukaryotic lineage of stramenopiles. Of the hundreds of diatom genera, two produce long fibers of chitin that extrude through their cell walls of silica. We identify and describe here genes encoding putative chitin synthases in a variety of additional diatom genera, indicating that the ability to produce chitin is more widespread and likely plays a more central role in diatom biology than previously considered. Diatom chitin synthases fall into four phylogenetic clades. Protein domain predictions and differential gene expression patterns provide evidence that chitin synthases have multiple functions within a diatom cell.
Thalassiosira pseudonana
possesses six genes encoding three types of chitin synthases. Transcript abundance of the gene encoding one of these chitin synthase types increases when cells resume division after short-term silicic acid starvation and during short-term limitation by silicic acid or iron, two nutrient conditions connected in the environment and known to affect the cell wall. During long-term silicic acid starvation transcript abundance of this gene and one additional chitin synthase gene increased at the same time a chitin-binding lectin localized to the girdle band region of the cell wall. Together, these results suggest that the ability to produce chitin is more widespread in diatoms than previously thought and that a subset of the chitin produced by diatoms is associated with the cell wall.
She3p Possesses a Novel Activity Required for
ASH1
mRNA Localization in
Saccharomyces cerevisiae
Authors: Landers Sharon M. , Gallas Michelle R. , Little Jaime , Long Roy M.
Published: May 2009

Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations within and between cells. One mechanism for sorting proteins is through RNA localization. In
Saccharomyces cerevisiae
,
ASH1
mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcriptional repressor Ash1p.
ASH1
mRNA localization requires four
cis
-acting localization elements and the
trans
-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directly transport
ASH1
mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the
ASH1
mRNA
cis
-acting elements. Currently, the role for She3p in
ASH1
mRNA localization is as an adaptor protein, since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants of She3p, S348E and the double mutant S343E S361E, that are defective for
ASH1
mRNA localization, and yet both of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest that She3p possesses a novel activity required for
ASH1
mRNA localization, and our data imply that this function is related to the ability of She3p to associate with
ASH1
mRNA. Interestingly, we determined that She3p is phosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sites of phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphorylation. The present study reveals that the current accepted model for
ASH1
mRNA localization does not fully account for the function of She3p in
ASH1
mRNA localization.
The Cell End Marker Protein TeaC Is Involved in Growth Directionality and Septation in
Aspergillus nidulans
Authors: Higashitsuji Yuhei , Herrero Saturnino , Takeshita Norio , Fischer Reinhard
Published: May 2009

Polarized growth in filamentous fungi depends on the correct spatial organization of the microtubule (MT) and actin cytoskeleton. In
Schizosaccharomyces pombe
it was shown that the MT cytoskeleton is required for the delivery of so-called cell end marker proteins, e.g., Tea1 and Tea4, to the cell poles. Subsequently, these markers recruit several proteins required for polarized growth, e.g., a formin, which catalyzes actin cable formation. The latest results suggest that this machinery is conserved from fission yeast to
Aspergillus nidulans.
Here, we have characterized TeaC, a putative homologue of Tea4. Sequence identity between TeaC and Tea4 is only 12.5%, but they both share an SH3 domain in the N-terminal region. Deletion of
teaC
affected polarized growth and hyphal directionality. Whereas wild-type hyphae grow straight, hyphae of the mutant grow in a zig-zag way, similar to the hyphae of
teaA
deletion (
tea1
) strains. Some small, anucleate compartments were observed. Overexpression of
teaC
repressed septation and caused abnormal swelling of germinating conidia. In agreement with the two roles in polarized growth and in septation, TeaC localized to hyphal tips and to septa. TeaC interacted with the cell end marker protein TeaA at hyphal tips and with the formin SepA at hyphal tips and at septa.
3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the
Sulfolobales
Authors: Teufel Robin , Kung Johannes W. , Kockelkorn Daniel , Alber Birgit E. , Fuchs Georg
Published: May 2009

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO
2
fixation in various
Crenarchaea
, as studied in some detail in
Metallosphaera sedula
. This cycle and the autotrophic 3-hydroxypropionate cycle in
Chloroflexus aurantiacus
have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In
M. sedula
the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from
M. sedula
or
Sulfolobus tokodaii
and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the
Metallosphaera
CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the
Sulfolobales
. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from
C. aurantiacus
, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in
Chloroflexus
and in the
Sulfolobales
evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.
Involvement of the Leucine Response Transcription Factor LeuO in Regulation of the Genes for Sulfa Drug Efflux
Authors: Shimada Tomohiro , Yamamoto Kaneyoshi , Ishihama Akira
Published: May 2009

LeuO, a LysR family transcription factor, exists in a wide variety of bacteria of the family
Enterobacteriaceae
and is involved in the regulation of as yet unidentified genes affecting the stress response and pathogenesis expression. Using genomic screening by systematic evolution of ligands by exponential enrichment (SELEX) in vitro, a total of 106 DNA sequences were isolated from 12 different regions of the
Escherichia coli
genome. All of the SELEX fragments formed complexes in vitro with purified LeuO. After Northern blot analysis of the putative target genes located downstream of the respective LeuO-binding sequence, a total of nine genes were found to be activated by LeuO, while three genes were repressed by LeuO. The LeuO target gene collection included several multidrug resistance genes. A phenotype microarray assay was conducted to identify the gene(s) responsible for drug resistance and the drug species that are under the control of the LeuO target gene(s). The results described herein indicate that the
yjcRQP
operon, one of the LeuO targets, is involved in sensitivity control against sulfa drugs. We propose to rename the
yjcRQP
genes the
sdsRQP
genes (
s
ulfa
d
rug
s
ensitivity determinant).
Gene Expression Patterns Associated with the Biosynthesis of the Sunscreen Scytonemin in
Nostoc punctiforme
ATCC 29133 in Response to UVA Radiation
Authors: Soule Tanya , Garcia-Pichel Ferran , Stout Valerie
Published: May 2009

Under exposure to UV radiation, some cyanobacteria synthesize sunscreen compounds. Scytonemin is a heterocyclic indole-alkaloid sunscreen, the synthesis of which is induced upon exposure to UVA (long-wavelength UV) radiation. We previously identified and characterized an 18-gene cluster associated with scytonemin biosynthesis in the cyanobacterium
Nostoc punctiforme
ATCC 29133; we now report on the expression response of these genes to a step-up shift in UVA exposure. Using quantitative PCR on cDNAs from the
N. punctiforme
transcriptome and primers targeting each of the 18 genes in the cluster, we followed their differential expression in parallel subcultures incubated with and without UVA. All 18 genes are induced by UVA irradiation, with relative transcription levels that generally peak after 48 h of continuous UVA exposure. A five-gene cluster implicated in the process of scytonemin biosynthesis solely on the basis of comparative genomics was also upregulated. Furthermore, we demonstrate that all of the genes in the18-gene region are cotranscribed as part of a single transcriptional unit.
The Crystal Structures of Substrate and Nucleotide Complexes of
Enterococcus faecium
Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily
Authors: Young Paul G. , Walanj Rupa , Lakshmi Vendula , Byrnes Laura J. , Metcalf Peter , Baker Edward N. , Vakulenko Sergei B. , Smith Clyde A.
Published: May 2009

Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in
Enterococcus
species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic
R
factor of 0.210 (
R
free
, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.
Gemfibrozil Inhibits
Legionella pneumophila
and
Mycobacterium tuberculosis
Enoyl Coenzyme A Reductases and Blocks Intracellular Growth of These Bacteria in Macrophages
Authors: Reich-Slotky Ronit , Kabbash Christina A. , Della-Latta Phyllis , Blanchard John S. , Feinmark Steven J. , Freeman Sherry , Kaplan Gilla , Shuman Howard A. , Silverstein Samuel C.
Published: May 2009

We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of
Legionella pneumophila
and of 27 strains of wild-type and multidrug-resistant
Mycobacterium tuberculosis
in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited
L. pneumophila
fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an
L. pneumophila
FabI enoyl reductase homolog that complemented for growth an
Escherichia coli
strain carrying a temperature-sensitive enoyl reductase and rendered the complemented
E. coli
strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this
L. pneumophila
FabI homolog, as well as
M. tuberculosis
InhA and
E. coli
FabI.
3′-Phosphoadenosine-5′-Phosphate Phosphatase Activity Is Required for Superoxide Stress Tolerance inStreptococcus mutans
Authors: Zhang Jiaqin , Biswas Indranil
Published: May 2009

Aerobic microorganisms have evolved different strategies to withstand environmental oxidative stresses generated by various reactive oxygen species (ROS). For the facultative anaerobic human oral pathogenStreptococcus mutans, the mechanisms used to protect against ROS are not fully understood, since it does not possess catalase, an enzyme that degrades hydrogen peroxide. In order to elucidate the genes that are essential for superoxide stress response, methyl viologen (MV)-sensitive mutants ofS. mutanswere generated via ISS1mutagenesis. Screening of approximately 2,500 mutants revealed six MV-sensitive mutants, each containing an insertion in one of five genes, including a highly conserved hypothetical gene, SMU.1297. Sequence analysis suggests that SMU.1297 encodes a hypothetical protein with a high degree of homology to theBacillus subtilisYtqI protein, which possesses an oligoribonuclease activity that cleaves nano-RNAs and a phosphatase activity that degrades 3′-phosphoadenosine-5′-phosphate (pAp) and 3′-phosphoadenosine-5′-phosphosulfate (pApS) to produce AMP; the latter activity is similar to the activity of theEscherichia coliCysQ protein, which is required for sulfur assimilation. SMU.1297 was deleted using a markerless Cre-loxP-based strategy; the SMU.1297 deletion mutant was just as sensitive to MV as the ISS1insertion mutant. Complementation of the deletion mutant with wild-type SMU.1297, intrans, restored the parental phenotype. Biochemical analyses with purified SMU.1297 protein demonstrated that it has pAp phosphatase activity similar to that of YtqI but apparently lacks an oligoribonuclease activity. The ability of SMU.1297 to dephosphorylate pApS in vivo was confirmed by complementation of anE. coli cysQmutant with SMU.1297 intrans. Thus, our results suggest that SMU.1297 is involved in superoxide stress tolerance inS. mutans. Furthermore, the distribution of homologs of SMU.1297 in streptococci indicates that this protein is essential for superoxide stress tolerance in these organisms.
Uptake of Glycerol-2-Phosphate via the
ugp
-Encoded Transporter in
Escherichia coli
K-12
Authors: Yang Kechao , Wang Mi , Metcalf William W.
Published: May 2009

During phenotypic characterization of various
Escherichia coli
mutants, we observed that Δ
phoA
strains are capable of using glycerol-2-phosphate (G2P) as a sole source of phosphorus. Mutations in the
ugpBAECQ
operon eliminated this phenotype, suggesting that G2P is a previously unrecognized substrate for the binding protein-dependent Ugp transporter.
orf4
of the
Bacillus cereus sigB
Gene Cluster Encodes a General Stress-Inducible Dps-Like Bacterioferritin
Authors: Wang Shin-Wei , Chen Chien-Yen , Tseng Joseph T. , Liang Shih-Hsiung , Chen Ssu-Ching , Hsieh Chienyan , Chen Yen-hsu , Chen Chien-Cheng
Published: May 2009

The function of
orf4
in the
sigB
cluster in
Bacillus cereus
ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an
orf4
-null mutant and produced recombinant protein rOrf4 to establish the role of
orf4
. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl
2
. Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of
orf4
had no effect on activation of the alternative sigma factor σ
B
, and therefore,
orf4
is not associated with σ
B
regulation; however,
orf4
can be significantly upregulated upon environmental stress but not H
2
O
2
treatment.
B. cereus
strains with constitutive Orf4 expression exhibited a viability higher than that of the
orf4
-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.
The
ldhA
Gene, Encoding Fermentative
l
-Lactate Dehydrogenase of
Corynebacterium glutamicum
, Is under the Control of Positive Feedback Regulation Mediated by LldR
Authors: Toyoda Koichi , Teramoto Haruhiko , Inui Masayuki , Yukawa Hideaki
Published: May 2009

Corynebacterium glutamicum ldhA
encodes
l
-lactate dehydrogenase, a key enzyme that couples
l
-lactate production to reoxidation of NADH formed during glycolysis. We previously showed that in the absence of sugar, SugR binds to the
ldhA
promoter region, thereby repressing
ldhA
expression. In this study we show that LldR is another protein that binds to the
ldhA
promoter region, thus regulating
ldhA
expression. LldR has hitherto been characterized as an
l
-lactate-responsive transcriptional repressor of
l
-lactate utilization genes. Transposon mutagenesis of a reporter strain carrying a chromosomal
ldhA
promoter-
lacZ
fusion (P
ldhA
-
lacZ
) revealed that
ldhA
disruption drastically decreased expression of P
ldhA
-
lacZ
. P
ldhA
-
lacZ
expression in the
ldhA
mutant was restored by deletion of
lldR
, suggesting that LldR acts as a repressor of
ldhA
in the absence of
l
-lactate and the LldR-mediated repression is not relieved in the
ldhA
mutant due to its inability to produce
l
-lactate.
lldR
deletion did not affect P
ldhA
-
lacZ
expression in the wild-type background during growth on either glucose, acetate, or
l
-lactate. However, it upregulated P
ldhA
-
lacZ
expression in the
sugR
mutant background during growth on acetate. The binding sites of LldR and SugR are located around the −35 and −10 regions of the
ldhA
promoter, respectively.
C. glutamicum ldhA
expression is therefore primarily repressed by SugR in the absence of sugar. In the presence of sugar, SugR-mediated repression of
ldhA
is alleviated, and
ldhA
expression is additionally enhanced by LldR inactivation in response to
l
-lactate produced by LdhA.
The
Sinorhizobium meliloti
LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa
Authors: Haag Andreas F. , Wehmeier Silvia , Beck Sebastian , Marlow Victoria L. , Fletcher Vivien , James Euan K. , Ferguson Gail P.
Published: May 2009

Free-living
Sinorhizobium meliloti lpxXL
and
acpXL
mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of
S. meliloti
bacteroid lipid A with VLCFAs.
Intact Flagellar Motor of
Borrelia burgdorferi
Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion
Authors: Liu Jun , Lin Tao , Botkin Douglas J. , McCrum Erin , Winkler Hanspeter , Norris Steven J.
Published: May 2009

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact
Borrelia burgdorferi
to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of
B. burgdorferi
, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained.
flgI
transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.
Site-Specific Recombination in the Cyanobacterium
Anabaena
sp. Strain PCC 7120 Catalyzed by the Integrase of Coliphage HK022
Authors: Melnikov Olga , Zaritsky Arieh , Zarka Aliza , Boussiba Sammy , Malchin Natalia , Yagil Ezra , Kolot Mikhail
Published: May 2009

The integrase (Int) of the λ-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host,
Escherichia coli
. Int recognizes two different pairs of recombining sites
attP
×
attB
and
attL
×
attR
for integration and excision, respectively. This system was adapted to the cyanobacterium
Anabaena
sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into
Anabaena
cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate

P
glnA

-
attL
-T1/T2-
attR
-
lacZ
, where T1/T2 are the strong transcription terminators of
rrnB
, to prevent expression of the
lacZ
reporter under the constitutive promoter

P
glnA

. The Int-catalyzed site-specific recombination reaction was monitored by the expression of
lacZ
emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in
Anabaena
cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host,
E. coli
.
Involvement of Two Transport Systems and a Specific Porin in the Uptake of Phthalate by
Burkholderia
spp
Authors: Chang Hung-Kuang , Dennis Jonathan J. , Zylstra Gerben J.
Published: May 2009

Burkholderia
spp. that degrade phthalate have an ABC transporter-type phthalate transport system (OphFGH) and a specific porin (OphP) in addition to a permease-type phthalate transporter (OphD). OphFGH has a lower
K

m

and higher
V
max
than OphD, which affects how the bacteria grow. OphP is involved in both mechanisms of transport.
High-Force Generation Is a Conserved Property of Type IV Pilus Systems
Authors: Clausen Martin , Jakovljevic Vladimir , Søgaard-Andersen Lotte , Maier Berenike
Published: May 2009

The type IV pilus (T4P) system of
Neisseria gonorrhoeae
is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in
Myxococcus xanthus
of 149 ± 14 pN exceeds the force generated by
N. gonorrhoeae
. Retraction velocities including a bimodal distribution were similar between
M. xanthus
and
N. gonorrhoeae
, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an
M. xanthus
mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 ± 10 pN. Thus,
M. xanthus
harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles.
Genome Sequence ofAzotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes
Authors: Setubal João C. , dos Santos Patricia , Goldman Barry S. , Ertesvåg Helga , Espin Guadelupe , Rubio Luis M. , Valla Svein , Almeida Nalvo F. , Balasubramanian Divya , Cromes Lindsey , Curatti Leonardo , Du Zijin , Godsy Eric , Goodner Brad , Hellner-Burris Kaitlyn , Hernandez José A. , Houmiel Katherine , Imperial Juan , Kennedy Christina , Larson Timothy J. , Latreille Phil , Ligon Lauren S. , Lu Jing , Mærk Mali , Miller Nancy M. , Norton Stacie , O'Carroll Ina P. , Paulsen Ian , Raulfs Estella C. , Roemer Rebecca , Rosser James , Segura Daniel , Slater Steve , Stricklin Shawn L. , Studholme David J. , Sun Jian , Viana Carlos J. , Wallin Erik , Wang Baomin , Wheeler Cathy , Zhu Huijun , Dean Dennis R. , Dixon Ray , Wood Derek
Published: May 2009

Azotobacter vinelandiiis a soil bacterium related to thePseudomonasgenus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence ofA. vinelandiiDJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes,A. vinelandiiis specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application ofA. vinelandiias a host for the production and characterization of oxygen-sensitive proteins.
Interactions of DNA with Biofilm-Derived Membrane Vesicles
Authors: Schooling Sarah R. , Hubley Amanda , Beveridge Terry J.
Published: May 2009

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.
Two-Step Assembly Dynamics of theBacillussubtilisDivisome
Authors: Gamba Pamela , Veening Jan-Willem , Saunders Nigel J. , Hamoen Leendert W. , Daniel Richard A.
Published: May 2009

Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly inBacillussubtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division inB.subtilisis mainly regulated at the posttranscriptional level.
Identification of ORF636 in Phage φSLT Carrying Panton-Valentine Leukocidin Genes, Acting as an Adhesion Protein for a Poly(Glycerophosphate) Chain of Lipoteichoic Acid on the Cell Surface of
Staphylococcus aureus
Authors: Kaneko Jun , Narita-Yamada Sachiko , Wakabayashi Yukari , Kamio Yoshiyuki
Published: May 2009

The temperate phage φSLT of
Staphylococcus aureus
carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of
S. aureus
. ORF636 bound specifically to
S. aureus
; it did not bind to any other staphylococcal species or to several gram-positive bacteria.