bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Tetracycline-Resistant
Streptococcus thermophilus
Isolates from Italian Soft Cheeses
Authors: Rizzotti Lucia , La Gioia Federica , Dellaglio Franco , Torriani Sandra
Published: April 2009

Tetracycline-resistant
Streptococcus thermophilus
isolates from soft cheeses harbored the genes
tet
(S),
tet
(M), and
tet
(L). Molecular analysis of these genes revealed their expression, localization on plasmids or Tn
916
-Tn
1545
family transposons, and their similarity with published sequences. The study highlights the importance of an accurate safety assessment of using
S. thermophilus
as a starter culture.
Widespread Abundance of Functional Bacterial Amyloid in Mycolata and Other Gram-Positive Bacteria
Authors: Jordal Peter Bruun , Dueholm Morten Simonsen , Larsen Poul , Petersen Steen Vang , Enghild Jan Johannes , Christiansen Gunna , Højrup Peter , Nielsen Per Halkjær , Otzen Daniel Erik
Published: April 2009

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, includingEscherichia coli,Salmonella, and the gram-positive organismStreptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phylaActinobacteriaandFirmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the generaStreptomyces,Bacillus, andNocardiawere all coated with amyloids. FuBA was purified fromGordonia amarae(from the cell envelope) andGeodermatophilus obscurus, and they had the morphology, tinctorial properties, and β-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope ofG. amaraewas visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.
Aldouronate Utilization in
Paenibacillus
sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism
Authors: Nong Guang , Rice John D. , Chow Virginia , Preston James F.
Published: April 2009

Paenibacillus
sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 α-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/β-xylosidase (XynB). Here we show that this
Paenibacillus
sp. strain is able to utilize methylglucuronoxylose (MeGAX
1
), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX
3
), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX
n
). The average rates of utilization of MeGAX
n
, MeGAX
1
, and MeGAX
3
were 149.8, 59.4, and 54.3 μg xylose equivalents·ml
−1
·h
−1
, respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX
1
and MeGAX
3
, releasing 4-
O
-methyl-
d
-glucuronate α-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX
3
, to xylose and xylobiose. The ability to utilize MeGAX
1
provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX
n
than that of MeGAX
3
indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the
aguA
and
xynA2
genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.
Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments
Authors: Jogler Christian , Lin Wei , Meyerdierks Anke , Kube Michael , Katzmann Emanuel , Flies Christine , Pan Yongxin , Amann Rudolf , Reinhardt Richard , Schüler Dirk
Published: April 2009

In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.
Phenotypic and Transcriptomic Analyses of Mildly and Severely Salt-Stressed
Bacillus cereus
ATCC 14579 Cells
Authors: den Besten Heidy M. W. , Mols Maarten , Moezelaar Roy , Zwietering Marcel H. , Abee Tjakko
Published: April 2009

Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen
Bacillus cereus
ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions.
B. cereus
continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na
+
/H
+
, and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms.
In Silico Prediction of Horizontal Gene Transfer Events in
Lactobacillus bulgaricus
and
Streptococcus thermophilus
Reveals Protocooperation in Yogurt Manufacturing
Authors: Liu Mengjin , Siezen Roland J. , Nauta Arjen
Published: April 2009

Lactobacillus bulgaricus
and
Streptococcus thermophilus
, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both
L. bulgaricus
and
S. thermophilus
. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from
S. thermophilus
to
L. bulgaricus
, and the gene cluster
cbs-cblB
(
cglB
)-
cysE
for the metabolism of sulfur-containing amino acids, transferred from
L. bulgaricus
or
Lactobacillus helveticus
to
S. thermophilus
. The HGT event for the
cbs-cblB
(
cglB
)-
cysE
gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.
Reverse Transcription of 16S rRNA To Monitor Ribosome-Synthesizing Bacterial Populations in the Environment
Authors: Lu Ting , Stroot Peter G. , Oerther Daniel B.
Published: April 2009

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3′ tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5′ tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of
Acinetobacter calcoaceticus
ATCC 23055
T
. Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.
Identification of the
Candida albicans
Cap1p Regulon
Authors: Znaidi Sadri , Barker Katherine S. , Weber Sandra , Alarco Anne-Marie , Liu Teresa T. , Boucher Geneviève , Rogers P. David , Raymond Martine
Published: April 2009

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in
Candida albicans
. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in
C. albicans
, we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA
3
) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA
3
) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA
3
). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA
3
or Cap1p-CSE-HA
3
(the binding ratio was at least twofold;
P
≤ 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3′ ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (
P
≤ 0.02) included 11 genes involved in the OSR (
CAP1
,
GLR1
,
TRX1
,
SOD1
,
CAT1
, and others), 13 genes involved in response to drugs (
PDR16
,
MDR1
,
FLU1
,
YCF1
,
FCR1
, and others), 4 genes involved in phospholipid transport (
PDR16
,
GIT1
,
RTA2
, and orf19.932), and 3 genes involved in the regulation of nitrogen utilization (
GST3
, orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5′-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.
Comparative Genomics Using Microarrays Reveals Divergence and Loss of Virulence-Associated Genes in Host-Specific Strains of the Insect PathogenMetarhizium anisopliae
Authors: Wang Sibao , Leclerque Andreas , Pava-Ripoll Monica , Fang Weiguo , St. Leger Raymond J.
Published: April 2009

Many strains ofMetarhizium anisopliaehave broad host ranges, but others are specialists and adapted to particular hosts. Patterns of gene duplication, divergence, and deletion in three generalist and three specialist strains were investigated by heterologous hybridization of genomic DNA to genes from the generalist strain Ma2575. As expected, major life processes are highly conserved, presumably due to purifying selection. However, up to 7% of Ma2575 genes were highly divergent or absent in specialist strains. Many of these sequences are conserved in other fungal species, suggesting that there has been rapid evolution and loss in specialistMetarhiziumgenomes. Some poorly hybridizing genes in specialists were functionally coordinated, indicative of reductive evolution. These included several involved in toxin biosynthesis and sugar metabolism in root exudates, suggesting that specialists are losing genes required to live in alternative hosts or as saprophytes. Several components of mobile genetic elements were also highly divergent or lost in specialists. Exceptionally, the genome of the specialist cricket pathogen Ma443 contained extra insertion elements that might play a role in generating evolutionary novelty. This study throws light on the abundance of orphans in genomes, as 15% of orphan sequences were found to be rapidly evolving in the Ma2575 lineage.
Induction of Follicular Gastritis following Postthymectomy Autoimmune Gastritis inHelicobacter pylori-Infected BALB/c Mice
Authors: Oshima Chikashi , Okazaki Kazuichi , Matsushima Yumi , Sawada Mitsutaka , Chiba Tsutomu , Takahashi Kiyoyuki , Hiai Hiroshi , Katakai Tomoya , Kasakura Shinpei , Masuda Tohru
Published: May 2009

Helicobacter pyloriis the major causative agent of chronic antral gastritis and is thought to be involved in the pathogenesis of mucosa-associated lymphoid tissue lymphoma (MALToma) developing in the human stomach. The aim of this study was to clarify whether corporal autoimmune gastritis (AIG), which is known to decrease acidity due to destruction of parietal cells, predisposes mice toH. pyloriinfection, thereby leading to MALToma-like pathology. BALB/c mice in which AIG had been induced by thymectomy 3 days after birth (AIG mice) were used. The AIG mice were orally administered mouse-adaptedH. pyloriat the age of 6 weeks and were examined histologically and serologically after 2 to 12 months. The results were compared with those obtained from uninfected AIG mice and infected normal mice. Germinal centers were induced in the corpus in 57% of theH. pylori-infected AIG mice, which elicited anti-H. pyloriantibody responses in association with upregulation of interleukin-4 (IL-4) mRNA. In these mice, parietal cells remained in the corpus mucosa. These findings were in contrast to those with the uninfected AIG mice: fundic gland atrophy due to disappearance of parietal cells associated with upregulation of gamma interferon, but not IL-4, mRNA and no germinal center formation in the corpus. These observations suggest that AIG alters the infectivity ofH. pylori, leading to MALToma-like follicular gastritis, at an early stage afterH. pyloriinfection.
Differential Binding of Clonal Variants ofPlasmodium falciparumto Allelic Forms of Intracellular Adhesion Molecule 1 Determined by Flow Adhesion Assay
Authors: Adams S. , Turner G. D. H. , Nash G. B. , Micklem K. , Newbold C. I. , Craig A. G.
Published: May 2009

Adhesion ofPlasmodium falciparum-infected erythrocytes to the endothelial ligand intercellular adhesion molecule 1 (ICAM-1) has been implicated in the pathogenesis of cerebral malaria. Recently, a high-frequency coding polymorphism in the N-terminal domain of ICAM-1 (ICAM-1Kilifi) that is associated with susceptibility to cerebral disease in Kenya has been described. Preliminary static adhesion assays suggested that two different selectedP. falciparumlines, ITO4-A4 (A4) and ItG-ICAM (ItG), have different properties of binding to the natural variant proteins ICAM-1 and ICAM-1Kilifi. Using a flow adhesion assay system, we have confirmed differences between the two lines in binding of parasitized erythrocytes to the variant ICAM-1 proteins. Total adhesion of ItG-infected erythrocytes to ICAM-1 and ICAM-1Kilifiis greater than that of A4-infected erythrocytes, and erythrocytes infected by both parasite strains show reduced binding to ICAM-1Kilifi. However, under these physiologically relevant flow conditions, we have shown differences between A4 and ItG strains in dynamic rolling behavior on ICAM-1Kilifi. The percentage of erythrocytes infected with A4 that roll on both ICAM-1 and ICAM-1Kilifiis greater than that of those infected with ItG. Also, the rolling velocity of A4-infected erythrocytes on ICAM-1Kilifiis markedly increased compared to that on ICAM-1, in contrast to the rolling velocity of ItG-infected erythrocytes, which is similar on both proteins. These findings suggest that different parasite lines can vary in their avidity for the same host ligand, which may have important consequences for the pathophysiology ofP. falciparummalaria.
Interleukin-12 as an Adjuvant Promotes Immunoglobulin G and Type 1 Cytokine Recall Responses to Major Surface Protein 2 of the Ehrlichial PathogenAnaplasma marginale
Authors: Tuo Wenbin , Palmer Guy H. , McGuire Travis C. , Zhu Daming , Brown Wendy C.
Published: May 2009

Anaplasma marginaleis a tick-transmitted pathogen of cattle closely related to the human ehrlichiae,Ehrlichia chaffeensisand the agent of human granulocytic ehrlichiosis (HGE). These pathogens have in common a structurally conserved outer membrane protein (OMP) designated the major surface protein 2 (MSP-2) inA. marginaleand HGE and OMP-1 inE. chaffeensis. Protective immunity against ehrlichial pathogens is believed to require induction of gamma interferon (IFN-γ) and opsonizing immunoglobulin (Ig) subclasses directed against OMP epitopes that, in concert, activate macrophages for phagocytosis and killing. Because interleukin-12 (IL-12) acts as an adjuvant for protein immunization to induce IFN-γ and protective immunity against intracellular pathogens, we hypothesized that as an adjuvant with MSP-2, IL-12 would augment type 1 recall responses toA. marginale. IL-12 was coadsorbed with MSP-2 to alum and shown to significantly enhance IFN-γ production by lymph node cells (LNC) and LNC-derived CD4+T-cell lines from immunized calves following recall stimulation withA. marginale. LNC proliferation and IL-2 production were also enhanced in IL-12-treated calves. Elevated recall proliferative responses by peripheral blood mononuclear cells were still evident 9 months after immunization. Serum IgG levels were consistently increased in IL-12 immunized calves, predominantly due to higher IgG1 responses. The results support the use of IL-12 coadsorbed with OMP of ehrlichial pathogens in alum to amplify both antibody and type-1 cytokine responses important for protective immunity.
Comparative Analysis of the Mucosal Adjuvanticity of the Type II Heat-Labile Enterotoxins LT-IIa and LT-IIb
Authors: Martin Michael , Metzger Daniel J. , Michalek Suzanne M. , Connell Terry D. , Russell Michael W.
Published: May 2009

Cholera toxin (CT) and the heat-labile enterotoxin ofEscherichia coli(LT-I) are members of the serogroup I heat-labile enterotoxins (HLT) and can serve as systemic and mucosal adjuvants. However, information is lacking with respect to the structurally related but antigenically distinct serogroup II HLT, LT-IIa and LT-IIb, which have different binding specificities for ganglioside receptors. The purpose of this study was to assess the effectiveness of LT-IIa and LT-IIb as mucosal adjuvants in comparison to the prototypical type I HLT, CT. BALB/c mice were immunized by the intranasal (i.n.) route with the surface protein adhesin AgI/II ofStreptococcus mutansalone or supplemented with an adjuvant amount of CT, LT-IIa, or LT-IIb. Antigen-specific antibody responses in saliva, vaginal wash, and plasma were assayed by enzyme-linked immunosorbent assay. Mice given AgI/II with LT-IIa or LT-IIb by the i.n. route had significantly higher mucosal and systemic antibody responses than mice immunized with AgI/II alone. Anti-AgI/II immunoglobulin A (IgA) antibody activity in saliva and vaginal secretions of mice given AgI/II with LT-IIa or LT-IIb was statistically similar in magnitude to that seen in mice given AgI/II and CT. LT-IIb significantly enhanced the number of AgI/II-specific antibody-secreting cells in the draining superficial cervical lymph nodes compared to LT-IIa and CT. LT-IIb and CT induced significantly higher plasma anti-AgI/II IgG titers compared to LT-IIa. When LT-IIb was used as adjuvant, the proportion of plasma IgG2a relative to IgG1 anti-AgI/II antibody was elevated in contrast to the predominance of IgG1 antibodies promoted by AgI/II alone or when CT or LT-IIa was used. In vitro stimulation of AgI/II-specific cells from the superficial lymph nodes and spleen revealed that LT-IIa and LT-IIb induced secretion of interleukin-4 and significantly higher levels of gamma interferon compared to CT. These results demonstrate that the type II HLT LT-IIa and LT-IIb exhibit potent and distinct adjuvant properties for stimulating immune responses to a noncoupled protein immunogen after mucosal immunization.
Isolation of
Bacteroides fragilis
Mutants with In Vivo Growth Defects by Using Tn
4400
′, a Modified Tn
4400
Transposition System, and a New Screening Method
Authors: Tang Yixin P. , Malamy Michael H.
Published: May 2009

A modified version of the
Bacteroides fragilis
transposon Tn
4400
, designated Tn
4400
′, enabling rapid isolation and analysis of
B. fragilis
mutants has been constructed. To identify potential virulence factors, Tn
4400
′-generated mutants were screened by a new method; this resulted in the isolation of 21 mutant strains with impaired growth characteristics on tissue culture monolayers but normal growth in rich medium anaerobically.
Episomal Expression of Specific Sense and Antisense mRNAs inLeishmania amazonensis: Modulation of gp63 Level in Promastigotes and Their Infection of Macrophages In Vitro
Authors: Chen De-Qiao , Kolli Bala Krishna , Yadava Nagendra , Lu Hong Gang , Gilman-Sachs Alice , Peterson Daniel A. , Chang Kwang-Poo
Published: May 2009

The major surface glycoprotein (gp63) ofLeishmania amazonensisis a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into aLeishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.
Identification of Genes Involved in Biofilm Formation and Respiration via Mini-
Himar
Transposon Mutagenesis of
Geobacter sulfurreducens
Authors: Rollefson Janet B. , Levar Caleb E. , Bond Daniel R.
Published: April 2009

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe
Geobacter sulfurreducens
requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in
Geobacter
spp. and identify novel genes involved in this process.
Escherichia coli
mating strains and plasmids were used to develop a conjugation protocol and deliver mini-
Himar
transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in
Geobacter
spp.
Characterization of the
Escherichia coli
ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System
Authors: Lien Hsiang-Yun , Shy Ru-Shan , Peng Sheng-Shiang , Wu Yuei-Long , Weng Yu-Ting , Chen Hsuan-He , Su Pin-Chih , Ng Wei-Fu , Chen Yu-Chun , Chang Pei-Yi , Wu Whei-Fen
Published: April 2009

In
Escherichia coli
, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and
cpsB
::
lacZ
assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.
Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria
Authors: Wischgoll Simon , Taubert Martin , Peters Franziska , Jehmlich Nico , von Bergen Martin , Boll Matthias
Published: April 2009

In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO
2
. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms
Geobacter metallireducens
(GDH
Geo
) (Fe[III] reducing) and
Desulfococcus multivorans
(GDH
Des
) (sulfate reducing). GDH
Geo
was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced
bamM
gene. The gene coding for GDH
Des
was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDH
Geo
catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDH
Des
catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDH
Geo
, the

K
m

was 30 ± 2 μM and the
V
max
was 3.2 ± 0.2 μmol min
−1
mg
−1
, and for GDH
Des
, the

K
m

was 52 ± 5 μM and the
V
max
was 11 ± 1 μmol min
−1
mg
−1
. GDH
Des
but not GDH
Geo
was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDH
Geo
but are missing in GDH
Des
. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.
Subcellular Localization of TatAd of
Bacillus subtilis
Depends on the Presence of TatCd or TatCy
Authors: Ridder Anja N. J. A. , de Jong Esther J. , Jongbloed Jan D. H. , Kuipers Oscar P.
Published: April 2009

The gram-positive bacterium
Bacillus subtilis
contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in
B. subtilis
is controlled by TatC.
The Quorum Sensing-Dependent Gene
katG
of
Burkholderia glumae
Is Important for Protection from Visible Light
Authors: Chun Heejin , Choi Okhee , Goo Eunhye , Kim Nayeon , Kim Hongsup , Kang Yongsung , Kim Jinwoo , Moon Jae Sun , Hwang Ingyu
Published: April 2009

Quorum sensing (QS) plays important roles in the pathogenicity of
Burkholderia glumae
, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in
B. glumae
. The QS-defective mutant of
B. glumae
exhibited less catalase activity than wild-type
B. glumae
. A β-glucuronidase assay of a
katG
::Tn
3-gusA78
reporter fusion protein revealed that
katG
expression is under the control of QS. Furthermore,
katG
expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates
katG
expression. The
katG
mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 μmol
−1
m
−2
, the survival rate of the
katG
mutant was 10
5
-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the
katG
mutant.
In Vivo Sensitivity of Blue-Light-Dependent Signaling Mediated by AppA/PpsR or PrrB/PrrA in
Rhodobacter sphaeroides
Authors: Metz Sebastian , Jäger Andreas , Klug Gabriele
Published: April 2009

Formation of photosynthesis complexes in
Rhodobacter sphaeroides
is regulated in a redox- and light-dependent manner by the AppA/PpsR and PrrB/PrrA systems. While on the one hand, blue light is sensed by the flavin adenine dinucleotide-binding BLUF domain of AppA, on the other, light is absorbed by bacteriochlorophyll signals through PrrB/PrrA. We show that much smaller quantities initiate the AppA-mediated response to blue light than the bacteriochlorophyll-mediated response.
ResDE-Dependent Regulation of Enterotoxin Gene Expression in
Bacillus cereus
: Evidence for Multiple Modes of Binding for ResD and Interaction with Fnr
Authors: Esbelin Julia , Armengaud Jean , Zigha Assia , Duport Catherine
Published: April 2009

In the food-borne pathogen
Bacillus cereus
F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of
hbl
and
nhe
transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (
p
) of the enterotoxin regulator genes
resDE
,
fnr
, and
plcR
and the enterotoxin structural genes
nhe
and
hbl
, but with different affinities. Binding of ResD to
pplcR
,
pnhe
, and
phbl
was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and
presDE
and
pfnr
. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates
B. cereus
virulence.
Molecular Dissection of the
secA2
Locus of Group B Streptococcus Reveals that Glycosylation of the Srr1 LPXTG Protein Is Required for Full Virulence
Authors: Mistou Michel-Yves , Dramsi Shaynoor , Brega Sara , Poyart Claire , Trieu-Cuot Patrick
Published: April 2009

In streptococci, the
secA2
locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The
secA2
locus of
Streptococcus agalactiae
strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.
Active Transcription of rRNA Operons Condenses the Nucleoid inEscherichia coli: Examining the Effect of Transcription on Nucleoid Structure in the Absence of Transertion
Authors: Cabrera Julio E. , Cagliero Cedric , Quan Selwyn , Squires Catherine L. , Jin Ding Jun
Published: April 2009

InEscherichia colithe genome must be compacted ∼1,000-fold to be contained in a cellular structure termed the nucleoid. It is proposed that the structure of the nucleoid is determined by a balance of multiple compaction forces and one major expansion force. The latter is mediated by transertion, a coupling of transcription, translation, and translocation of nascent membrane proteins and/or exported proteins. In supporting this notion, it has been shown consistently that inhibition of transertion by the translation inhibitor chloramphenicol results in nucleoid condensation due to the compaction forces that remain active in the cell. Our previous study showed that during optimal growth, RNA polymerase is concentrated into transcription foci or “factories,” analogous to the eukaryotic nucleolus, indicating that transcription and RNA polymerase distribution affect the nucleoid structure. However, the interpretation of the role of transcription in the structure of the nucleoid is complicated by the fact that transcription is implicated in both compacting forces and the expansion force. In this work, we used a new approach to further examine the effect of transcription, specifically from rRNA operons, on the structure of the nucleoid, when the major expansion force was eliminated. Our results showed that transcription is necessary for the chloramphenicol-induced nucleoid compaction. Further, an active transcription from multiple rRNA operons in chromosome is critical for the compaction of nucleoid induced by inhibition of translation. All together, our data demonstrated that transcription of rRNA operons is a key mechanism affecting genome compaction and nucleoid structure.
Autoproteolysis of YscU of
Yersinia pseudotuberculosis
Is Important for Regulation of Expression and Secretion of Yop Proteins
Authors: Björnfot Ann-Catrin , Lavander Moa , Forsberg Åke , Wolf-Watz Hans
Published: April 2009

YscU of
Yersinia
can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU
CC
. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-
cis
-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca
2+
conditions) and calcium-depleted medium (−Ca
2+
conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca
2+
-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca
2+
conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca
2+
conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.
Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in
Streptococcus sanguinis
Authors: Das Sankar , Kanamoto Taisei , Ge Xiuchun , Xu Ping , Unoki Takeshi , Munro Cindy L. , Kitten Todd
Published: April 2009

Streptococcus sanguinis
is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in
S. sanguinis
strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (
lgt
) and signal peptidase II (
lspA
). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in
lgt
,
lspA
, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of
ssaB
, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [
3
H]palmitate labeling and Western blot analysis confirmed that the
lgt
mutant failed to acylate lipoproteins, that the
lspA
mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.
Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in
Shewanella oneidensis
MR-1
Authors: Charania M. A. , Brockman K. L. , Zhang Y. , Banerjee A. , Pinchuk G. E. , Fredrickson J. K. , Beliaev A. S. , Saffarini D. A.
Published: April 2009

Unlike other bacteria that use FNR to regulate anaerobic respiration,
Shewanella oneidensis
MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes,
cyaA
,
cyaB
, and
cyaC
, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an
Escherichia coli cya
mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either
cyaA
or
cyaB
did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of
cyaC
caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a
crp
mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both
cyaA
and
cyaC
were deleted. Microarray analysis of gene expression in the
crp
and
cyaC
mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the
cyaC
mutant but not in the
crp
mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.
Evidence on the effects of exercise therapy in the treatment of chronic disease
Authors: Kujala U M
Published: May 2009

Evidence on the effects of exercise in the treatment of patients with chronic diseases should be based on well designed randomised controlled trials. The most consistent finding of the meta-analyses summarised in the present work is that aerobic/functional capacity and muscle strength can be improved by exercise training among patients with different diseases without having detrimental effects on disease progression. This is important, as with population aging exercise therapy may be an important means of reducing disability and increasing the number of older people living independently. Additionally, there is accumulating evidence that in patients with chronic disease exercise therapy is effective in improving the prognostic risk factor profile and, in certain diseases, in delaying mortality. In some diseases, such as osteoarthritis, pain symptoms may also be reduced. Severe complications during the exercise therapy programs were rare.
NanoMORALs — Metal nanoparticles doped with organic molecules
Authors: Palmisano Giovanni , Augugliaro Vincenzo , Ciriminna Rosaria , Pagliaro Mario
Published: May 2009

A new class of nanosized metal–organic alloys (MORALs) has been synthesized for the first time. Silver nanoparticles doped with Cu(II) and Fe(III) phthalocyanines were thus synthesized in the presence of sodium dodecyl sulfate (SDS). The resulting materials were characterized by means of XRD, SEM, TEM coupled to energy dispersive X-ray analysis, and thermogravimetric analysis. No leaching of the photoactive dopant species was observed in water or in common organic solvents.