bluuebunny/crossref_metadata_2025 huggingface dataset:
Subsurface Microbial Diversity in Deep-Granitic-Fracture Water in Colorado
Authors: Sahl Jason W. , Schmidt Raleigh , Swanner Elizabeth D. , Mandernack Kevin W. , Templeton Alexis S. , Kieft Thomas L. , Smith Richard L. , Sanford William E. , Callaghan Robert L. , Mitton Jeffry B. , Spear John R.
Published: November 2007

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (
Gallionella
spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that
Nitrosomonadales
dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by
Firmicutes
,
Proteobacteria
, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This “Henderson candidate division” dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions
Proteobacteria
(primarily the family
Ralstoniaceae
) and
Firmicutes
. Sequences grouping within
Ralstoniaceae
were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.
Mutations in the
lux
Operon of Natural Dark Mutants in the Genus
Vibrio
Authors: O'Grady Elizabeth A. , Wimpee Charles F.
Published: November 2007

Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess
lux
genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus
Vibrio
. Screening of over 600
Vibrio
isolates with a
luxA
gene probe revealed that approximately 5% carried the
luxA
gene. Bioluminescence assays of the
luxA
-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS
10
insertion sequence in
luxA
, a mutated
luxE
stop codon, and a truncated
luxH
. Strain BCB494 has a 396-bp deletion in
luxC
, and strain BCB440 has a frameshift in
luxC
. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete
lux
operons in natural isolates.
Formation of
N
-Ethylmaleimide (NEM)-Glutathione Conjugate and
N
-Ethylmaleamic Acid Revealed by Mass Spectral Characterization of Intracellular and Extracellular Microbial Metabolites of NEM
Authors: Mojica Elmer-Rico E. , Kim Sungpyo , Aga Diana S.
Published: November 2007

The extracellular and intracellular metabolites formed upon exposure of activated sludge microorganisms to a sublethal concentration of
N
-ethylmaleimide were monitored by liquid chromatography with ion trap mass spectrometry. The metabolite
N
-ethylsuccinimido-
S
-glutathione (
m
/
z
433) was converted rapidly to
N
-(2-oxoethyl)-2,2-(propionylamino)propanamide (
m
/
z
187) and
N
-ethylmaleamic acid (
m
/
z
144).
Short-Term Temporal Variability in Airborne Bacterial and Fungal Populations
Authors: Fierer Noah , Liu Zongzhi , Rodríguez-Hernández Mari , Knight Rob , Henn Matthew , Hernandez Mark T.
Published: November 2007

Airborne microorganisms have been studied for centuries, but the majority of this research has relied on cultivation-dependent surveys that may not capture all of the microbial diversity in the atmosphere. As a result, our understanding of airborne microbial ecology is limited despite the relevance of airborne microbes to human health, various ecosystem functions, and environmental quality. Cultivation-independent surveys of small-subunit rRNA genes were conducted in order to identify the types of airborne bacteria and fungi found at a single site (Boulder, CO) and the temporal variability in the microbial assemblages over an 8-day period. We found that the air samples were dominated by ascomycete fungi of the
Hypocreales
order and a diverse array of bacteria, including members of the proteobacterial and
Cytophaga-Flavobacterium-Bacteroides
groups that are commonly found in comparable culture-independent surveys of airborne bacteria. Bacterium/fungus ratios varied by 2 orders of magnitude over the sampling period, and we observed large shifts in the phylogenetic diversity of bacteria present in the air samples collected on different dates, shifts that were not likely to be related to local meteorological conditions. We observed more phylogenetic similarity between bacteria collected from geographically distant sites than between bacteria collected from the same site on different days. These results suggest that outdoor air may harbor similar types of bacteria regardless of location and that the short-term temporal variability in airborne bacterial assemblages can be very large.
Species of Environmental Mycobacteria Differ in Their Abilities To Grow in Human, Mouse, and Carp Macrophages and with Regard to the Presence of Mycobacterial Virulence Genes, as Observed by DNA Microarray Hybridization
Authors: Harriff Melanie J. , Wu Martin , Kent Michael L. , Bermudez Luiz E.
Published: November 2007

There are many species of environmental mycobacteria (EM) that infect animals that are important to the economy and research and that also have zoonotic potential. The genomes of very few of these bacterial species have been sequenced, and little is known about the molecular mechanisms by which most of these opportunistic pathogens cause disease. In this study, 18 isolates of EM isolated from fish and humans (including strains of
Mycobacterium avium
,
Mycobacterium peregrinum
,
Mycobacterium chelonae
, and
Mycobacterium salmoniphilum
) were examined for their abilities to grow in macrophage lines from humans, mice, and carp. Genomic DNA from 14 of these isolates was then hybridized against DNA from an
M. avium
reference strain, with a custom microarray containing virulence genes of mycobacteria and a selection of representative genes from metabolic pathways. The strains of EM had different abilities to grow within the three types of cell lines, which grouped largely according to the host from which they were isolated. Genes identified as being putatively absent in some of the strains included those with response regulatory functions, cell wall compositions, and fatty acid metabolisms as well as a recently identified pathogenicity island important to macrophage uptake. Further understanding of the role these genes play in host specificity and pathogenicity will be important to gain insight into the zoonotic potential of certain EM as well as their mechanisms of virulence.
Fitness Variation of
Borrelia burgdorferi
Sensu Stricto Strains in Mice
Authors: Hanincová Klára , Ogden Nicholas H. , Diuk-Wasser Maria , Pappas Christopher J. , Iyer Radha , Fish Durland , Schwartz Ira , Kurtenbach Klaus
Published: November 2007

Lyme borreliosis in North America is caused by the tick-borne spirochete
Borrelia burgdorferi
, a zoonotic bacterium that is able to persistently infect a wide range of vertebrate species. Given the pronounced strain structure of
B. burgdorferi
in the northeastern United States, we asked whether the fitness of the different genotypes varies among susceptible vertebrate hosts. The transmission dynamics of two genetically divergent human isolates of
B. burgdorferi
, BL206 and B348, were analyzed experimentally in white-footed mice and in C3H/HeNCrl mice over a time period of almost 3 months. We found that the initially high transmission efficiency from white-footed mice to ticks declined sharply for isolate B348 but remained considerably high for isolate BL206. In contrast, in C3H/HeNCrl mice, high transmission efficiency persisted for both isolates. Our findings provide proof-of-principle evidence for intrinsic fitness variation of
B. burgdorferi
strains in vertebrate host species, perhaps indicating the beginnings of adaptive radiation.
Growth of Thermophilic and Hyperthermophilic Fe(III)-Reducing Microorganisms on a Ferruginous Smectite as the Sole Electron Acceptor
Authors: Kashefi Kazem , Shelobolina Evgenya S. , Elliott W. Crawford , Lovley Derek R.
Published: November 2007

Recent studies have suggested that the structural Fe(III) within phyllosilicate minerals, including smectite and illite, is an important electron acceptor for Fe(III)-reducing microorganisms in sedimentary environments at moderate temperatures. The reduction of structural Fe(III) by thermophiles, however, has not previously been described. A wide range of thermophilic and hyperthermophilicArchaeaandBacteriafrom marine and freshwater environments that are known to reduce poorly crystalline Fe(III) oxides were tested for their ability to reduce structural (octahedrally coordinated) Fe(III) in smectite (SWa-1) as the sole electron acceptor. Two out of the 10 organisms tested,Geoglobus ahangariandGeothermobacterium ferrireducens, were not able to conserve energy to support growth by reduction of Fe(III) in SWa-1 despite the fact that both organisms were originally isolated with solid-phase Fe(III) as the electron acceptor. The other organisms tested were able to grow on SWa-1 and reduced 6.3 to 15.1% of the Fe(III). This is 20 to 50% less than the reported amounts of Fe(III) reduced in the same smectite (SWa-1) by mesophilic Fe(III) reducers. Two organisms,Geothermobacter ehrlichiiand archaeal strain 140, produced copious amounts of an exopolysaccharide material, which may have played an active role in the dissolution of the structural iron in SWa-1 smectite. The reduction of structural Fe(III) in SWa-1 by archaeal strain 140 was studied in detail. Microbial Fe(III) reduction was accompanied by an increase in interlayer and octahedral charges and some incorporation of potassium and magnesium into the smectite structure. However, these changes in the major element chemistry of SWa-1 smectite did not result in the formation of an illite-like structure, as reported for a mesophilic Fe(III) reducer. These results suggest that thermophilic Fe(III)-reducing organisms differ in their ability to reduce and solubilize structural Fe(III) in SWa-1 smectite and that SWa-1 is not easily transformed to illite by these organisms.
Utilization of Molasses Sugar for Lactic Acid Production by
Lactobacillus delbrueckii
subsp.
delbrueckii
Mutant Uc-3 in Batch Fermentation
Authors: Dumbrepatil Arti , Adsul Mukund , Chaudhari Shivani , Khire Jayant , Gokhale Digambar
Published: November 2007

Efficient lactic acid production from cane sugar molasses by
Lactobacillus delbrueckii
mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.
(Per)chlorate Reduction by the Thermophilic BacteriumMoorella perchloratireducenssp. nov., Isolated from Underground Gas Storage
Authors: Balk Melike , van Gelder Ton , Weelink Sander A. , Stams Alfons J. M.
Published: November 2007

A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 μm in diameter and 2 to 8 μm in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70°C, with an optimum at 55 to 60°C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter−1with an optimum at 10 g liter−1. Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related toMoorella thermoaceticaandMoorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.
In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging
Authors: Walczysko Petr , Kuhlicke Ute , Knappe Sabine , Cordes Christiana , Neu Thomas R.
Published: November 2007

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio inEscherichia colicells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate ofE. colicells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.
Comparison and Validation of Methods To Quantify Cry1Ab Toxin from
Bacillus thuringiensis
for Standardization of Insect Bioassays
Authors: Crespo André L. B. , Spencer Terence A. , Nekl Emily , Pusztai-Carey Marianne , Moar William J. , Siegfried Blair D.
Published: November 2007

Standardization of toxin preparations derived from
Bacillus thuringiensis
(Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer,
Ostrinia nubilalis
(Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of
O. nubilalis
. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC
50
values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.
Thickness and Surface Density of Extracellular Polymers on
Acidithiobacillus ferrooxidans
Authors: Taylor Eric S. , Lower Steven K.
Published: November 2007

In vivo force microscopy measurements of
Acidithiobacillus ferrooxidans
revealed a repulsive force that was due to the presence of extracellular polymers on the bacterium's surface. Measured force-distance profiles were fit to steric force theory to estimate the density and thickness values of these exopolymers. The polymer densities were 3.4 × 10
16
to 7.1 × 10
16
molecules m
−2
, and the equilibrium thickness was 29 nm.
Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic
Escherichia coli
O157:NM
Authors: Mellmann Alexander , Lu Shan , Karch Helge , Xu Jian-guo , Harmsen Dag , Schmidt M. Alexander , Bielaszewska Martina
Published: November 2007

Using colony blot hybridization with
stx
2
and
eae
probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated
stx
2
-positive and
eae
-positive sorbitol-fermenting (SF) enterohemorrhagic
Escherichia coli
(EHEC) O157:NM (nonmotile) strains from initial stool specimens and
stx
-negative and
eae
-positive SF
E. coli
O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The
stx
-negative isolates from each patient shared with the corresponding
stx
2
-positive isolates
fliC
H7
, non-
stx
virulence traits, and multilocus sequence types, which indicates that they arose from the
stx
2
-positive strains by loss of
stx
2
during infection. Analysis of the integrity of the
yecE
gene, a possible
stx
phage integration site in EHEC O157, in the consecutive
stx
2
-positive and
stx
-negative isolates demonstrated that
yecE
was occupied in
stx
2
-positive but intact in
stx
-negative strains. It was possible to infect and lysogenize the
stx
-negative
E. coli
O157 strains in vitro using an
stx
2
-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the
stx
2
-containing phage resulted in the occupation of
yecE
and production of biologically active Shiga toxin 2. We conclude that the
yecE
gene in SF
E. coli
O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their
stx
-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of
stx
2
-harboring phages. The ability to recycle
stx
2
, a critical virulence trait, makes SF
E. coli
O157:NM strains ephemeral EHEC that can exist as
stx
-negative variants during certain phases of their life cycle.
Carbon Source Dependence and Photostimulation of Conidiation in
Hypocrea atroviridis
Authors: Friedl Martina A. , Kubicek Christian P. , Druzhinina Irina S.
Published: November 2007

Hypocrea atroviridis
is frequently used as a photomorphogenetic model due to its ability to conidiate upon exposure to light. Light is thereby believed to be the primary trigger for spore formation. In contrast, we show here that conidiation is primarily carbon source dependent and that illumination plays a catalytic role; of a total of 95 tested carbon sources, only a small set of carbohydrates, polyols, and sugar acids allowed conidiation in darkness, and on most of them, conidiation was significantly more strongly expressed in light. In addition, there are also a number of carbon sources on which
H. atroviridis
conidiates in darkness, but light does not further stimulate the process. Yet on another small set of carbon sources (
l
-sorbitol,
d
-fucose,
d
- and
l
-arabinose, and erythritol),
H. atroviridis
shows better sporulation in darkness than in light. No sporulation was observed on organic acids and amino acids. Mutants with deletions in the two blue-light receptor proteins BLR-1 and BLR-2 generally showed weaker conidiation on a smaller number of carbon sources than did the parental strain, yet they clearly sporulated on 15 and 27 of the 95 carbon sources tested, respectively. Of the carbon sources supporting sporulation, only 11 supported the conidiation of both mutants, suggesting that the BLR-1 and BLR-2 receptors are variously involved in the carbon source-dependent regulation of spore formation. The addition of cyclic AMP, which has been reported to lead to conidiation in darkness, both positively and negatively affected sporulation and resulted in different effects in the parental strain and the two Δ
blr
mutants. Our data show that the carbon source is the prime determinant for conidiation and that it influences the organism's regulation of conidiation by means of BLR-1 and BLR-2 and their cross talk with cyclic AMP.
Characterization of Plasmacytoid Dendritic Cells in Bone Marrow of Pig-Tailed Macaques
Authors: Reeves R. Keith , Fultz Patricia N.
Published: November 2007

Plasmacytoid dendritic cells (pDCs), one of two types of bone marrow (BM)-derived blood DCs, play an important role in linking innate and adaptive immune responses. However, little is known about the nature of pDCs that reside in the BM. Because the simian immunodeficiency virus-macaque model closely mimics human immunodeficiency virus disease in humans, with both infections inducing a decrease in pDCs, we characterized and compared pDCs in the BM with those in peripheral blood (PB) of healthy pig-tailed macaques. The results revealed that pDCs from both compartments had the same CD123++HLA-DR+Lin−phenotype and were similar in size. Although BM-derived pDCs (BM-pDCs) were 3-fold greater in frequency and 10-fold greater in number, they had lower cell surface expression of both HLA-DR and the costimulatory molecule CD86 than did PB-pDCs. Both BM- and PB-pDCs responded ex vivo to synthetic CpG oligodeoxynucleotides and inactivated influenza virus by upregulating HLA-DR and CD86 and secreting cytokines; however, stimulated BM-pDCs secreted less alpha interferon and tumor necrosis factor alpha per cell than did PB-pDCs. These results suggest that while BM-pDCs appear to be phenotypically less mature than PB-pDCs, they do respond to pathogens. Thus, during acute infections, these cells could initiate immune responses either in the BM or after rapidly migrating from the BM into the periphery. A better characterization of pDCs in blood and tissues will be beneficial for future studies of macaques that focus on either pathogenesis or vaccine development.
Mycobacterium bovisBacillus Calmette-Guérin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-κB and -Jun N-Terminal Kinase Signaling Pathways
Authors: Méndez-Samperio Patricia , Trejo Artemisa , Pérez Aline
Published: November 2007

In response toMycobacterium bovisbacillus Calmette-Guérin (BCG), CC chemokines are secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) has been shown to recognizeM. bovisBCG and to initiate signaling pathways that result in enhanced secretion of CC chemokines. Despite the essential requirement of TLR2 inM. bovisBCG infection, the mechanisms by which it induces secretion of CC chemokines are not well defined. In this study, we report that stimulation of HEK293 cells expressing human TLR2 withM. bovisBCG resulted in increased CCL2 and CCL5 secretion, as determined by an enzyme-linked immunosorbent assay.M. bovisBCG infection resulted in the activation of c-Jun N-terminal kinase (JNK), and the inhibition of JNK activity had a significant effect onM. bovisBCG-dependent CCL5 secretion in TLR2-expressing cells but no effect onM. bovisBCG-dependent CCL2 secretion from infected HEK293 cells expressing human TLR2. TheM. bovisBCG-induced CCL5 release was attenuated by sulfasalazine (a well-described inhibitor of NF-κB activity), BAY 11-7082 (an IκB phosphorylation inhibitor), and ALLN (a well-described inhibitor of NF-κB activation that prevents degradation of IκB and eventually results in a lack of translocated NF-κB in the nucleus). In addition, stimulation of TLR2-expressing cells withM. bovisBCG resulted in translocation of NF-κB subunits from the cytoplasmic to the nuclear fraction, and stimulation of cells withM. bovisBCG activated IκB kinase αβ. These findings indicate thatM. bovisBCG induces CCL5 production through mechanisms that include a TLR2-dependent component that requires JNK and NF-κB activities.
Elucidating the Role of C/D snoRNA in rRNA Processing and Modification inTrypanosoma brucei
Authors: Barth Sarit , Shalem Boaz , Hury Avraham , Tkacz Itai Dov , Liang Xue-hai , Uliel Shai , Myslyuk Inna , Doniger Tirza , Salmon-Divon Mali , Unger Ron , Michaeli Shulamit
Published: November 2007

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2′-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA inTrypanosoma bruceiare fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2′-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.
Morphogenesis Control in
Candida albicans
and
Candida dubliniensis
through Signaling Molecules Produced by Planktonic and Biofilm Cells
Authors: Martins Margarida , Henriques Mariana , Azeredo Joana , Rocha Sílvia M. , Coimbra Manuel A. , Oliveira Rosário
Published: November 2007

Morphogenesis control by chemical signaling molecules is beginning to be highlighted in
Candida
biology. The present study focuses on morphogenic compounds produced in situ by
Candida albicans
and
Candida dubliniensis
during planktonic and biofilm growth that may at least partially substantiate the effect promoted by supernatants in morphogenesis. For both species, planktonic versus biofilm supernatants were analyzed by headspace-solid-phase microextraction and gas chromatography-mass spectrometry. Both planktonic cells and biofilm supernatants of
C. albicans
and
C. dubliniensis
contained isoamyl alcohol, 2-phenylethanol, 1-dodecanol,
E
-nerolidol, and
E
,
E
-farnesol. Alcohol secretion profiles were species, culture mode, and growth time specific. The addition of exogenous alcohols to the cultures of both species inhibited the morphological transition from the yeast to the filamentous form by up to 50%. The physiological role of these alcohols was put to evidence by comparing the effects of a 96-h cultured supernatant with synthetic mixtures containing isoamyl alcohol, 2-phenylethanol,
E
-nerolidol, and
E
,
E
-farnesol at concentrations determined herein. All synthetic mixtures elicited a morphological effect similar to that observed for the corresponding supernatants when used to treat
C. albicans
and
C. dubliniensis
cultures, except for the effect of the 96-h
C. dubliniensis
planktonic supernatant culture on
C. albicans
. Overall, these results reveal a group of alcohol extracellular signaling molecules that are biologically active with
C. albicans
and
C. dubliniensis
morphogenesis.
Novel 44-Kilodalton Subunit of Axonemal Dynein Conserved from
Chlamydomonas
to Mammals
Authors: Yamamoto Ryosuke , Yanagisawa Haru-aki , Yagi Toshiki , Kamiya Ritsu
Published: November 2007

Cilia and flagella have multiple dyneins in their inner and outer arms.
Chlamydomonas
inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett.
580
:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the
ida4
and
ida5
mutants, both lacking this dynein. In the
ida5
axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.
Regulation of Rot Expression in
Staphylococcus aureus
Authors: Hsieh Hsin-Yeh , Tseng Ching Wen , Stewart George C.
Published: November 2007

Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in
Staphylococcus aureus
. The function of Rot, but not the transcription of
rot
, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (σ
B
) has a repressive effect on
rot
expression during the postexponential phase of growth. The transcriptional profiles of Rot in σ
B
-positive and σ
B
-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of
rot
expression was observed during the stationary phase of growth, and this upregulation occurred in a σ
B
-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the
rot
promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of
rot
expression was also observed in
sarS
-negative strains. Affinity purification of proteins binding to the
rot
promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the
rot
promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the
rot
mRNA but not the inhibition of
rot
transcription.
Agrobacterium tumefaciens
C58 Uses ActR and FnrN To Control
nirK
and
nor
Expression
Authors: Baek Seung-Hun , Hartsock Angela , Shapleigh James P.
Published: November 2007

Agrobacterium tumefaciens
can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator
actR
significantly reduced
nirK
expression and Nir activity but not
nnrR
expression. Purified ActR bound to the
nirK
promoter but not the
nor
or
nnrR
promoter. A putative ActR binding site was identified in the
nirK
promoter region using mutational analysis and an in vitro binding assay. A
nirK
promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter
lacZ
fusion. Truncation of the
nirK
promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of
paz
, encoding the copper protein pseudoazurin, was highly reduced in the
actR
or
fnrN
mutants and that ActR binds to the
paz
promoter. Inactivation of
paz
reduced Nir activity by 55%. These results help explain why Nir activity is very low in the
actR
mutant even though a
nirK
promoter with mutations in the ActR binding site showed significant expression.
Characterization of the Saframycin A Gene Cluster fromStreptomyces lavendulaeNRRL 11002 Revealing a Nonribosomal Peptide Synthetase System for Assembling the Unusual Tetrapeptidyl Skeleton in an Iterative Manner
Authors: Li Lei , Deng Wei , Song Jie , Ding Wei , Zhao Qun-Fei , Peng Chao , Song Wei-Wen , Tang Gong-Li , Liu Wen
Published: November 2007

Saframycin A (SFM-A), produced byStreptomyces lavendulaeNRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.
Autoinducer-2-Regulated Genes inStreptococcus mutansUA159 and Global Metabolic Effect of theluxSMutation
Authors: Sztajer Helena , Lemme André , Vilchez Ramiro , Schulz Stefan , Geffers Robert , Yip Cindy Ying Yin , Levesque Celine M. , Cvitkovitch Dennis G. , Wagner-Döbler Irene
Published: November 2007

Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of aluxSnull mutant culture ofStreptococcus mutansUA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to aluxSnull mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the δ subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to theluxSmutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.
Protein-Protein Interactions within Type III Secretion System-Dependent Pili of
Rhizobium
sp. Strain NGR234
Authors: Saad Maged M. , Staehelin Christian , Broughton William J. , Deakin William J.
Published: November 2007

Pili synthesized by the type III secretion system of
Rhizobium
species strain NGR234 are essential for protein secretion and thus for efficient symbiosis with many legumes. Isolation and partial purification of these pili showed that they are composed of at least three proteins, NopA, NopB, and NopX. Using biochemical assays, we show here that these proteins interact directly with one another.
Nisin-Triggered Activity of Lys44, the Secreted Endolysin from
Oenococcus oeni
Phage fOg44
Authors: Nascimento João Gil , Guerreiro-Pereira Maria Carolina , Costa Sérgio Fernandes , São-José Carlos , Santos Mário Almeida
Published: November 2007

The intrinsic resistance of
Oenococcus oeni
cells to the secreted endolysin from oenophage fOg44 (Lys44) was investigated. Experiments with several antimicrobials support the hypothesis that the full activity of Lys44 requires sudden ion-nonspecific dissipation of the proton motive force, an event undertaken by the fOg44 holin in the phage infection context.
Clp-Dependent Proteolysis Down-Regulates Central Metabolic Pathways in Glucose-StarvedBacillus subtilis
Authors: Gerth Ulf , Kock Holger , Kusters Ilja , Michalik Stephan , Switzer Robert L. , Hecker Michael
Published: November 2007

Entry into stationary phase inBacillus subtilisis linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using35S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced inclpP,clpC, andclpXmutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.
Growth of
Streptococcus pneumoniae
on Human Glycoconjugates Is Dependent upon the Sequential Activity of Bacterial Exoglycosidases
Authors: Burnaugh Amanda M. , Frantz Laura J. , King Samantha J.
Published: November 2007

In the human host,
Streptococcus pneumoniae
encounters a variety of glycoconjugates, including mucin, host defense molecules, and glycans associated with the epithelial surface.
S. pneumoniae
is known to encode a number of glycosidases that may modify these glycoconjugates in vivo. Three exoglycosidases, a neuraminidase (NanA), β-galactosidase (BgaA), and
N
-acetylglucosaminidase (StrH), have been previously demonstrated to sequentially deglycosylate N-linked glycans on host defense molecules, which coat the pneumococcal surface in vivo. This cleavage is proposed to alter the clearance function of these molecules, allowing pneumococci to persist in the airway. However, we propose that the exoglycosidase-dependent liberation of monosaccharides from these glycoconjugates in close proximity to the pneumococcal surface provides
S. pneumoniae
with a convenient source of fermentable carbohydrate in vivo. In this study, we demonstrate that
S. pneumoniae
is able to utilize complex N-linked human glycoconjugates as a sole source of carbon to sustain growth and that efficient growth is dependent upon the sequential deglycosylation of the glycoconjugate substrate by pneumococcal exoglycosidases. In addition to demonstrating a role for NanA, BgaA, and StrH, we have identified a function for the second pneumococcal neuraminidase, NanB, in the deglycosylation of host glycoconjugates and have demonstrated that NanB activity can partially compensate for the loss or dysfunction of NanA. To date, all known functions of pneumococcal neuraminidase have been attributed to NanA. Thus, this study describes the first proposed role for NanB by which it may contribute to
S. pneumoniae
colonization and pathogenesis.
Cell Wall Carbohydrate Compositions of Strains from the
Bacillus cereus
Group of Species Correlate with Phylogenetic Relatedness
Authors: Leoff Christine , Saile Elke , Sue David , Wilkins Patricia , Quinn Conrad P. , Carlson Russell W. , Kannenberg Elmar L.
Published: November 2007

Members of the
Bacillus cereus
group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of
B. cereus
group members by separating them into clades and lineages. Based on MLST, we selected several
B. anthracis
,
B. cereus
, and
B. thuringiensis
strains and compared their cell wall carbohydrates. The cell walls of different
B. anthracis
strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal),
N
-acetyl mannosamine (ManNAc), and
N
-acetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (
B. cereus
type strain ATCC 14579 and
B. thuringiensis
strains) lacked Gal and contained
N
-acetylgalactosamine (GalNAc). The
B. cereus
clade 1 strains had cell walls that were similar in composition to
B. anthracis
in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in
B. anthracis
cell walls. Three recently identified clade 1 strains of
B. cereus
that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the
B. anthracis
strains. It was also observed that
B. anthracis
strains cell wall glycosyl compositions differed from one another in a plasmid-dependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of
atxA
, a global regulatory gene, from a pXO2
−
strain resulted in cell walls with an even greater level of Glc.
The NtrC Family Regulator AlgB, Which Controls Alginate Biosynthesis in MucoidPseudomonas aeruginosa, Binds Directly to thealgDPromoter
Authors: Leech Andrew J. , Sprinkle April , Wood Lynn , Wozniak Daniel J. , Ohman Dennis E.
Published: November 2007

Alginate production in mucoid (MucA-defective)Pseudomonas aeruginosais dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e.,algDoperon) activation. However, it remained unclear whether AlgB stimulatedalgDtranscription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1mucAbackground that overlapped with genes induced byd-cycloserine, which is known to activatealgDexpression. This set contained only thealgDoperon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to thealgDpromoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgDbut did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgDfragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp −224 to −274 relative to the start of PalgDtranscription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than σ54, in this case to stimulate transcription from the σ22-dependent PalgDpromoter.
A Mycobacterial
smc
Null Mutant Is Proficient in DNA Repair and Long-Term Survival
Authors: Güthlein Carolin , Wanner Roger M. , Sander Peter , Böttger Erik C. , Springer Burkhard
Published: November 2007

SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
defective in SMC were constructed. Surprisingly, inactivation of
smc
did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for
smc
null mutants in other species.