bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Sorting of the
Saccharomyces cerevisiae
Vacuolar Protein Sna4p
Authors: Pokrzywa W. , Guerriat B. , Dodzian J. , Morsomme P.
Published: January 2009

Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plants and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVBs). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic dileucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway by using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine, and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localize to both the vacuolar membrane and the lumen.
Killed but Metabolically Active
Bacillus anthracis
Vaccines Induce Broad and Protective Immunity against Anthrax
Authors: Skoble Justin , Beaber John W. , Gao Yi , Lovchik Julie A. , Sower Laurie E. , Liu Weiqun , Luckett William , Peterson Johnny W. , Calendar Richard , Portnoy Daniel A. , Lyons C. Rick , Dubensky Thomas W.
Published: January 2009

Bacillus anthracis
is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing
B. anthracis
that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the
spoIIE
and
uvrAB
genes, rendering
B. anthracis
extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the
lef
and
cya
genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA
B. anthracis
vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA
B. anthracis
-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA
B. anthracis
fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA
B. anthracis
were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.
Impaired Innate and Adaptive Immunity to
Streptococcus pneumoniae
and Its Effect on Colonization in an Infant Mouse Model
Authors: Bogaert Debby , Weinberger Daniel , Thompson Claudette , Lipsitch Marc , Malley Richard
Published: January 2009

Streptococcus pneumoniae
colonization and invasive disease peak around the third and first birthdays, respectively, and decline thereafter. While these declines are attributable in part to immunity acquired via natural exposure, maturation of innate immune responses may also be involved. A mucosally administered candidate whole-cell pneumococcal vaccine (WCV) containing killed pneumococcal antigen (WCA) plus a cholera toxin adjuvant protects against intranasal carriage of pneumococci by a mechanism that is antibody independent and CD4
+
TH17 cell dependent. Because infants and children are a key target population for this vaccine, we sought to evaluate the immune responses of neonatal and infant mice to
S. pneumoniae
and to assess whether the WCV would be effective in these mice. Like human infants, infant mice showed impaired clearance of nasopharyngeal colonization with
S. pneumoniae
. Macrophages from neonatal and infant mice stimulated with killed pneumococci in vitro showed significantly reduced cytokine production, including that of KC, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage chemoattractant protein 1, interleukin-6 (IL-6), IL-1α, tumor necrosis factor alpha, and gamma interferon, whereas IL-10 expression was significantly increased compared to that in macrophages from adult mice. IL-17A production from adult immune CD4
+
T cells was significantly delayed when neonatal macrophages instead of adult macrophages were used as antigen-presenting cells. Moreover, whole blood from mice immunized as neonates with WCV produced significantly less IL-17A after stimulation with WCA than did blood from mice immunized as adults. Nonetheless, a single immunization of neonatal mice with WCV significantly reduced colonization density. Overall, our data suggest an impairment of both innate and acquired cellular immune responses in neonatal and infant mice. However, WCV confers a significant reduction in colonization following pneumococcal challenge, suggesting that it may still be effective in the setting of immature immune responses.
Role of Deoxyribose Catabolism in Colonization of the Murine Intestine by PathogenicEscherichia coliStrains
Authors: Martinez-Jéhanne Vanessa , du Merle Laurence , Bernier-Fébreau Christine , Usein Codruta , Gassama-Sow Amy , Wane Abdul-Aziz , Gouali Malika , Damian Maria , Aïdara-Kane Awa , Germani Yves , Fontanet Arnaud , Coddeville Bernadette , Guérardel Yann , Le Bouguénec Chantal
Published: January 2009

We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by thedeoKoperon, is associated with the pathogenic potential ofEscherichia colistrains. Carbohydrate metabolism is thought to provide the nutritional support required forE. colito colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenicE. colistrains. Molecular and biochemical characterization of 1,221E. coliclones from various collections showed this biochemical trait to be common in theE. colispecies (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizingE. coliclones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenicE. coliwere significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of thedeoKoperon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenicE. coligrowth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.
Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway
Authors: Hasegawa Ayako , Sogo L. Farah , Tan Ming , Sütterlin Christine
Published: January 2009

Chlamydiais an obligate intracellular bacterium that grows and replicates inside a cytoplasmic inclusion. We report that a host protein, CD59, which regulates complement function at the surfaces of uninfected cells, can be detected at the membrane of the chlamydial inclusion. This localization to the inclusion membrane was specific for CD59 and not a general feature of other glycosylphosphatidylinositol (GPI)-anchored proteins or representative cell surface proteins. Using differential permeabilization studies, we showed that CD59 is localized to the luminal but not the cytoplasmic face of the inclusion membrane, consistent with membrane association via its GPI anchor. Furthermore, CD59 was present at the inclusion even when we prevented it from associating with membrane microdomains via the GPI anchor or when we inhibited general protein transport to the cell surface, indicating that a conventional Golgi apparatus-dependent trafficking mechanism was not involved. Based on these findings, we propose that selected host proteins are trafficked to the inclusion by a Golgi apparatus-independent pathway during aChlamydiainfection.
Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge withStreptococcus pneumoniaein Mice
Authors: Tian Haijun , Weber Sarah , Thorkildson Peter , Kozel Thomas R. , Pirofski Liise-anne
Published: January 2009

Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity toStreptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 μg of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcγ receptor IIB knockout (FcγRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcγRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcγR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcγR(s), whereas 5F6 and 7A9 required the inhibitory FcγR (FcγRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.
Direct Regulation by theVibrio choleraeRegulator ToxT To Modulate Colonization and Anticolonization Pilus Expression
Authors: Hsiao Ansel , Xu Xiao , Kan Biao , Kulkarni Rahul V. , Zhu Jun
Published: January 2009

The pathogenVibrio choleraeuses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host and establish itself at its preferred colonization site at the host intestinal mucosa. The key regulator in this process is the AraC/XylS family transcription factor, ToxT, which plays critical roles in pathogenesis, including the regulation of two type IV pili, the anticolonization factor mannose-sensitive hemagglutinin and the toxin-coregulated pilus. Previously, it was thought ToxT required dimerization in order to effect transcriptional regulation at its cognate promoters. Here, we present evidence that ToxT directly represses transcription of themshoperon by binding to three promoters within this operon and that dimerization may not be required for transcriptional repression of target promoters by ToxT, suggesting that this regulator uses different mechanisms to modulate the transcriptional repertoire ofV. cholerae.
Pseudomonas aeruginosa
Infection of Zebrafish Involves both Host and Pathogen Determinants
Authors: Clatworthy Anne E. , Lee Jenny See-Wai , Leibman Mark , Kostun Zachary , Davidson Alan J. , Hung Deborah T.
Published: January 2009

Zebrafish (
Danio rerio
) have a number of strengths as a host model for infection, including genetic tractability, a vertebrate immune system similar to that of mammals, ease and scale of laboratory handling, which allows analysis with reasonable throughput, and transparency, which facilitates visualization of the infection. With these advantages in mind, we examined whether zebrafish could be used to study
Pseudomonas aeruginosa
pathogenesis and found that infection of zebrafish embryos with live
P. aeruginosa
(PA14 or PAO1) by microinjection results in embryonic death, unlike infection with
Escherichia coli
or heat-killed
P. aeruginosa
, which has no effect. Similar to studies with mice,
P. aeruginosa
mutants deficient in type three secretion (
pscD
) or quorum sensing (
lasR
and
mvfR
) are attenuated in zebrafish embryos infected at 50 h postfertilization (hpf), a developmental stage when both macrophages and neutrophils are present. In contrast, embryos infected at 28 hpf, when only macrophages are initially present, succumb to lethal challenge with far fewer
P. aeruginosa
cells than those required for embryos infected at 50 hpf, are susceptible to infection with
lasR
and
pscD
deletion mutants, and are moderately resistant to infection with an
mvfR
mutant. Finally, we show that we can control the outcome of infection through the use of morpholinos, which allow us to shift immune cell numbers, or small molecules (antibiotics), which rescue embryos from lethal challenge. Thus, zebrafish are a novel host model that is well suited for studying the interactions among individual pathogenic functions of
P. aeruginosa
, the role of individual components of host immune defense, and small-molecule modulators of infection.
Salmonella enterica
Serovar Typhimurium Mutants Unable To Convert Malate to Pyruvate and Oxaloacetate Are Avirulent and Immunogenic in BALB/c Mice
Authors: Mercado-Lubo Regino , Leatham Mary P. , Conway Tyrrell , Cohen Paul S.
Published: January 2009

Previously, we showed that the
Salmonella enterica
serovar Typhimurium SR-11 tricarboxylic acid (TCA) cycle must operate as a complete cycle for full virulence after oral infection of BALB/c mice (M. Tchawa Yimga, M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen, Infect. Immun.
74:
1130-1140, 2006). In the same study, we showed that for full virulence, malate must be converted to both oxaloacetate and pyruvate. Moreover, it was recently demonstrated that blocking conversion of succinyl-coenzyme A to succinate attenuates serovar Typhimurium SR-11 but does not make it avirulent; however, blocking conversion of succinate to fumarate renders it completely avirulent and protective against subsequent oral infection with the virulent serovar Typhimurium SR-11 wild-type strain (R. Mercado-Lubo, E. J. Gauger, M. P. Leatham, T. Conway, and P. S. Cohen, Infect. Immun.
76:
1128-1134, 2008). Furthermore, the ability to convert succinate to fumarate appeared to be required only after serovar Typhimurium SR-11 became systemic. In the present study, evidence is presented that serovar Typhimurium SR-11 mutants that cannot convert fumarate to malate or that cannot convert malate to both oxaloacetate and pyruvate are also avirulent and protective in BALB/c mice. These results suggest that in BALB/c mice, the malate that is removed from the TCA cycle in serovar Typhimurium SR-11 for conversion to pyruvate must be replenished by succinate or one of its precursors, e.g., arginine or ornithine, which might be available in mouse phagocytes.
Burkholderia mallei tssMEncodes a Putative Deubiquitinase That Is Secreted and Expressed inside Infected RAW 264.7 Murine Macrophages
Authors: Shanks John , Burtnick Mary N. , Brett Paul J. , Waag David M. , Spurgers Kevin B. , Ribot Wilson J. , Schell Mark A. , Panchal Rekha G. , Gherardini Frank C. , Wilkinson Keith D. , DeShazer David
Published: January 2009

Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. TheB. malleiVirAG two-component regulatory system activates the transcription of ∼60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). TheB. mallei tssMgene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in avirAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. ThetssMgene was expressed within 1 h after uptake ofB. malleiinto RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provideB. malleia selective advantage in the intracellular environment during infection.
The RgpA-Kgp Proteinase-Adhesin Complexes ofPorphyromonas gingivalisInactivate the Th2 Cytokines Interleukin-4 and Interleukin-5
Authors: Tam Vivian , O'Brien-Simpson Neil M. , Chen Yu-Yen , Sanderson Colin J. , Kinnear Beverley , Reynolds Eric C.
Published: January 2009

The RgpA-Kgp proteinase-adhesin complexes are a primary virulence factor ofPorphyromonas gingivalis, a major pathogen in the development of chronic periodontitis. The RgpA-Kgp complexes have been suggested to bias the immune response to a Th2 phenotype in disease by hydrolysis of Th1 cytokines. Here, we show that the RgpA-Kgp complexes hydrolyze and inactivate interleukin-4 (IL-4) and IL-5 under physiologically relevant conditions. Using the IL-4/IL-5-dependent TF1.8 T-cell line, it was found that at equimolar ratios of cytokine to RgpA-Kgp complexes, IL-4 and IL-5 were inactivated in the culture medium. The inactivation of IL-4 and IL-5 was RgpA-Kgp concentration dependent, as at an enzyme-to-cytokine molar ratio of 1:8, the bioactivity of the cytokines was greater than at the higher concentration of RgpA-Kgp of 1:1. Furthermore, inactivation of the cytokines by the RgpA-Kgp complexes was time dependent, as longer preincubation times resulted in lower cytokine activity. IL-5 was found to be slightly more resistant to inactivation than IL-4. Mass spectrometric analyses of IL-4 and IL-5 showed that hydrolysis by RgpA-Kgp complexes was C terminal to Arg and Lys residues of the cytokines. The peptides released indicated that the regions of IL-4 and IL-5 important for bioactivity were being hydrolyzed in the first 15 min of incubation. The ability of the RgpA-Kgp complexes to degrade Th2 cytokines may contribute to immune dysregulation and may play a role in the pathology of chronic periodontitis.
Preferential Brain Homing following Intranasal Administration ofTrypanosoma cruzi
Authors: Caradonna Kacey , PereiraPerrin Mercio
Published: January 2009

The Chagas’ disease parasiteTrypanosoma cruzicommonly infects humans through skin abrasions or mucosa from reduviid bug excreta. Yet most studies on animal models start with subcutaneous or intraperitoneal injections, a distant approximation of the skin abrasion route. We show here that atraumatic placement ofT. cruziin the mouse nasal cavity produced low parasitemia, high survival rates, and preferential brain invasion compared to the case with subcutaneously injected parasites. Brain invasion was particularly prominent in the basal ganglia, peaked at a time when parasitemia was no longer detectable, and elicited a relatively large number of inflammatory foci. Yet, based on motor behavioral parameters and staining with Fluoro-Jade C, a dye that specifically recognizes apoptotic and necrotic neurons, brain invasion did not cause neurodegenerative events, in contrast to the neurodegeneration in the enteric nervous system. The results indicate that placement ofT. cruzion the mucosa in the mouse nasal cavity establishes a systemic infection with a robust yet harmless infection of the brain, seemingly analogous to disease progression in humans. The model may facilitate studies designed to understand mechanisms underlyingT. cruziinfection of the central nervous system.
Vaccination Route That Induces Transforming Growth Factor β Production Fails To Elicit Protective Immunity againstLeishmania donovaniInfection
Authors: Bhowmick Sudipta , Mazumdar Tuhina , Ali Nahid
Published: January 2009

BALB/c mice immunized intraperitoneally (i.p.) and intravenously (i.v.) withLeishmania donovanipromastigote membrane antigens (LAg), either free or encapsulated in liposomes, were protected against challenge infection withL. donovani, whereas mice immunized by the subcutaneous (s.c.) and intramuscular routes were not protected. Protected mice showed strong parasite resistance in both the liver and spleen, along with enhanced immunoglobulin G2a and delayed-type hypersensitivity responses. Again, mice vaccinated through the i.p. and i.v. routes showed high levels of NO production after challenge infection. s.c. vaccination resulted in an increased capacity of the spleen cells to produce prechallenge transforming growth factor β (TGF-β) levels during the in vitro antigen recall response, whereas i.p. immunization induced production of prechallenge gamma interferon, interleukin-12 (IL-12), and IL-4 levels, with a Th1 bias. Exposure to antigen-stimulated splenocyte supernatants of i.p. but not s.c. immunized mice activated macrophages for in vitro parasite killing. As an enhanced level of TGF-β was detected in supernatants from unprotected s.c. immunized mice, neutralization by anti-TGF-β antibody enhanced in vitro macrophage killing activity. The suppressive role of this cytokine was evaluated in vivo by vaccination with liposomal LAg and anti-TGF-β antibody. Upon parasite challenge, these animals showed significant protection in both the liver and spleen. Moreover, the addition of recombinant TGF-β in splenocyte supernatants of i.p. immunized mice in vitro as well as in vivo inhibited the protective ability of the macrophages by the i.p. route. Thus, the induction of high prechallenge TGF-β limits the efficacy of vaccination by routes that are nonprotective.
Complete Genome Sequence ofErythrobacter litoralisHTCC2594
Authors: Oh Hyun-Myung , Giovannoni Stephen J. , Ferriera Steve , Johnson Justin , Cho Jang-Cheon
Published: January 2009

Erythrobacter litoralishas been known as a bacteriochlorophylla-containing, aerobic, anoxygenic, phototrophic bacterium. Here we announce the complete genome sequence ofE. litoralisHTCC2594, which is devoid of phototrophic potential.E. litoralisHTCC2594, isolated by dilution-to-extinction culturing from seawater, could not carry out aerobic anoxygenic phototrophy and lacked genes for bacteriochlorophyllabiosynthesis and photosynthetic reaction center proteins.
Genetic Analysis of the Invariant Residue G791 in
Escherichia coli
16S rRNA Implicates RelA in Ribosome Function
Authors: Kim Hong-Man , Ryou Sang-Mi , Song Woo-Seok , Sim Se-Hoon , Cha Chang-Jun , Han Seung Hyun , Ha Nam-Chul , Kim Jae-Hong , Bae Jeehyeon , Cunningham Philip R. , Lee Kangseok
Published: January 2009

Previous studies identified G791 in
Escherichia coli
16S rRNA as an invariant residue for ribosome function. In order to establish the functional role of this residue in protein synthesis, we searched for multicopy suppressors of the mutant ribosomes that bear a G-to-U substitution at position 791. We identified
relA
, a gene whose product has been known to interact with ribosomes and trigger a stringent response. Overexpression of RelA resulted in the synthesis of approximately 1.5 times more chloramphenicol acetyltransferase (CAT) protein than could be synthesized by the mutant ribosomes in the absence of RelA overexpression. The ratio of mutant rRNA to the total ribosome pool was not changed, and the steady-state level of CAT mRNA was decreased by RelA overexpression. These data confirmed that the phenotype of RelA as a multicopy suppressor of the mutant ribosome did not result from the enhanced synthesis of mutant rRNA or CAT mRNA from the plasmid. To test whether the phenotype of RelA was related to the stringent response induced by the increased cellular level of (p)ppGpp, we screened for mutant RelA proteins whose overexpression enhances CAT protein synthesis by the mutant ribosomes as effectively as wild-type RelA overexpression and then screened for those whose overexpression does not produce sufficiently high levels of (p)ppGpp to trigger the stringent response under the condition of amino acid starvation. Overexpression of the isolated mutant RelA proteins resulted in the accumulation of (p)ppGpp in cells, which was amounted to approximately 18.2 to 38.9% of the level of (p)ppGpp found in cells that overexpress the wild-type RelA. These findings suggest that the function of RelA as a multicopy suppressor of the mutant ribosome does not result from its (p)ppGpp synthetic activity. We conclude that RelA has a previously unrecognized role in ribosome function.
Mutational Analysis and Homology-Based Modeling of the IntDOT Core-Binding Domain
Authors: Malanowska Karolina , Cioni Joel , Swalla Brian M. , Salyers Abigail , Gardner Jeffrey F.
Published: January 2009

Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.
Three-Dimensional Macromolecular Organization of CryofixedMyxococcus xanthusBiofilms as Revealed by Electron Microscopic Tomography
Authors: Palsdottir Hildur , Remis Jonathan P. , Schaudinn Christoph , O'Toole Eileen , Lux Renate , Shi Wenyuan , McDonald Kent L. , Costerton J. William , Auer Manfred
Published: January 2009

Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in thezaxis has opened a window in studies ofMyxococcus xanthuscell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.
The
Thermotoga maritima
Trk Potassium Transporter—from Frameshift to Function
Authors: Johnson Hope A. , Hampton Eric , Lesley Scott A.
Published: January 2009

The gene for the
Thermotoga maritima
Trk potassium transporter component TrkA was originally thought to be a frameshift mutation and not to encode a functional protein. However, expression from this gene yielded a complex consisting of two distinct proteins designated TM1088A and -B. Genetic complementation of
Escherichia coli
mutants unable to transport potassium suggests that TM1088A/B is part of a functional Trk potassium transporter complex with the membrane protein TM1089. The protein structure for TM1088A shows a characteristic Rossmann fold indicating an NAD
+
binding site and has structural similarity to potassium channel-related proteins. Ligand binding studies indicated that ATP, ADP, and AMP stabilized TM1088A to a much greater degree than NADH and NAD, consistent with the crystal structure of TM1088A, which contains a bound AMP natural ligand at the characteristic GXGXXG nucleotide binding site. Mutation of single and all glycines at this nucleotide binding site eliminated in vitro protein stabilization by the ligand, yet these mutated proteins could still functionally complement the
E. coli
potassium uptake mutants. We predict that this new two-subunit class of TrkA proteins is present in a number of organisms. A further subclass of the predicted two-subunit TrkA proteins lack an identifiable membrane-spanning subunit of the Trk K
+
transporter. This class, as exemplified by
Mycobacterium tuberculosis
, did not complement
E. coli
potassium transport with the native
E. coli
TrkH; thus, it may require a novel TrkH-like protein for activity or provide an alternate function in vivo.
Psychrobacter arcticus
273-4 Uses Resource Efficiency and Molecular Motion Adaptations for Subzero Temperature Growth
Authors: Bergholz Peter W. , Bakermans Corien , Tiedje James M.
Published: January 2009

Permafrost soils are extreme environments that exert low-temperature, desiccation, and starvation stress on bacteria over thousands to millions of years. To understand how
Psychrobacter arcticus
273-4 survived for >20,000 years in permafrost, transcriptome analysis was performed during growth at 22°C, 17°C, 0°C, and −6°C using a mixed-effects analysis of variance model. Genes for transcription, translation, energy production, and most biosynthetic pathways were downregulated at low temperatures. Evidence of isozyme exchange was detected over temperature for
d
-alanyl-
d
-alanine carboxypeptidases (
dac1
and
dac2
), DEAD-box RNA helicases (
csdA
and Psyc_0943), and energy-efficient substrate incorporation pathways for ammonium and acetate. Specific functions were compensated by upregulation of genes at low temperature, including genes for the biosynthesis of proline, tryptophan, and methionine. RNases and peptidases were generally upregulated at low temperatures. Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of a resource efficiency response. In contrast to results observed for other psychrophiles and mesophiles, only
clpB
and
hsp33
were upregulated at low temperature, and there was no upregulation of other chaperones and peptidyl-prolyl isomerases.
relA
,
csdA
, and
dac2
knockout mutants grew more slowly at low temperature, but a
dac1
mutant grew more slowly at 17°C. The combined data suggest that the basal biological machinery, including translation, transcription, and energy metabolism, is well adapted to function across the growth range of
P. arcticus
from −6°C to 22°C, and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.
Roles of
Agrobacterium tumefaciens
RirA in Iron Regulation, Oxidative Stress Response, and Virulence
Authors: Ngok-Ngam Patchara , Ruangkiattikul Nantaporn , Mahavihakanont Aekkapol , Virgem Susan S. , Sukchawalit Rojana , Mongkolsuk Skorn
Published: January 2009

The analysis of genetics and physiological functions of
Agrobacterium tumefaciens
RirA (rhizobial iron regulator) has shown that it is a transcription regulator and a repressor of iron uptake systems. The
rirA
mutant strain (NTLrirA) overproduced siderophores and exhibited a highly constitutive expression of genes involved in iron uptake (
fhuA
,
irp6A
, and
fbpA
) compared to that of the wild-type strain (NTL4). The deregulation in the iron control of iron uptake in NTLrirA led to iron overload in the cell, which was supported by the observation that the NTLrirA mutant was more sensitive than wild-type NTL4 to an iron-activated antibiotic, streptonigrin. The NTLrirA mutant was more sensitive than the parental strain to oxidants, including hydrogen peroxide, organic hydroperoxide, and a superoxide generator, menadione. However, the addition of an iron chelator, 2,2′-dipyridyl, reversed the mutant hypersensitivity to H
2
O
2
and organic hydroperoxide, indicating the role of iron in peroxide toxicity. Meanwhile, the reduced level of superoxide dismutase (SodBIII) was partly responsible for the menadione-sensitive phenotype of the NTLrirA mutant. The NTLrirA mutant showed a defect in tumorigenesis on tobacco leaves, which likely resulted from the increased sensitivity of NTLrirA to oxidants and the decreased ability of NTLrirA to induce virulence genes (
virB
and
virE
). These data demonstrated that RirA is important for
A. tumefaciens
during plant-pathogen interactions.
Pneumolysin Localizes to the Cell Wall of
Streptococcus pneumoniae
Authors: Price Katherine E. , Camilli Andrew
Published: January 2009

Streptococcus pneumoniae
is the causative agent of multiple diseases, including otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolytic pore-forming toxins, is produced by virtually all clinical isolates of
S. pneumoniae
, and strains in which the Ply gene has been deleted are severely attenuated in mouse models of infection. In contrast to all other members of the cholesterol-dependent cytolysin family, Ply lacks a signal peptide for export. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains ill defined. We determined by use of cell fractionation and Western blotting that, during in vitro growth, exported Ply is localized primarily to the cell wall compartment in 18 different serotypes in the absence of detectable cell lysis. Hemolytic assays revealed that this cell wall-localized Ply is active. Additionally, cell wall-localized Ply is accessible to extracellular protease and is detergent releasable.
The
Pseudomonas aeruginosa
Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis
Authors: Damron F. Heath , Qiu Dongru , Yu Hongwei D.
Published: January 2009

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in
Pseudomonas aeruginosa
is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of
P. aeruginosa
have a wild-type (wt)
mucA
. Here, we describe a loss-of-function mutation in
kinB
that causes overproduction of alginate in the wt
mucA
strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of
kinB
was correlated with high expression at the alginate-related promoters P

algU

and P

algD

. Deletion of alternative σ factor RpoN (σ
54
) or the response regulator AlgB in
kinB
mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the
kinB algB
double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when
kinB
was inactivated. The inactivation of the DegS-like protease AlgW in the
kinB
mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the
kinB
mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required
algB
and
rpoN
in the
kinB
mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt
mucA
strain PAO1.
The Sac10b Homolog in
Methanococcus maripaludis
Binds DNA at Specific Sites
Authors: Liu Yuchen , Guo Li , Guo Rong , Wong Richard L. , Hernandez Hilda , Hu Jinchuan , Chu Yindi , Amster I. Jonathan , Whitman William B. , Huang Li
Published: January 2009

The Sac10b protein family, also known as Alba, is widely distributed in
Archaea
. Sac10b homologs in thermophilic
Sulfolobus
species are very abundant. They bind both DNA and RNA with high affinity and without sequence specificity, and their physiological functions are still not fully understood. Mma10b from the euryarchaeote
Methanococcus maripaludis
is a mesophilic member of the Sac10b family. Mma10b is not abundant and constitutes only ∼0.01% of the total cellular protein. Disruption of mma10b resulted in poor growth of the mutant in minimal medium at near the optimal growth temperature but had no detectable effect on growth in rich medium. Quantitative proteomics, real time reverse transcription-PCR, and enzyme assays revealed that the expression levels of some genes involved in CO
2
assimilation and other activities were changed in the Δmma10b mutant. Chromatin immunoprecipitation suggested a direct association of Mma10b with an 18-bp DNA binding motif in vivo. Electrophoretic mobility shift assays and DNase I footprinting confirmed that Mma10b preferentially binds specific sequences of DNA with an apparent
K

d

in the 100 nM range. These results suggested that the physiological role of Mma10b in the mesophilic methanococci is greatly diverged from that of homologs in thermophiles.
Copper Acquisition Is Mediated by YcnJ and Regulated by YcnK and CsoR in
Bacillus subtilis
Authors: Chillappagari Shashi , Miethke Marcus , Trip Hein , Kuipers Oscar P. , Marahiel Mohamed A.
Published: January 2009

Copper is an essential cofactor for many enzymes, and at over a threshold level, it is toxic for all organisms. To understand the mechanisms underlying copper homeostasis of the gram-positive bacterium
Bacillus subtilis
, we have performed microarray studies under copper-limiting conditions. These studies revealed that the
ycnJ
gene encodes a protein that plays an important role in copper metabolism, as it shows a significant, eightfold upregulation under copper-limiting conditions and its disruption causes a growth-defective phenotype under copper deprivation as well as a reduced intracellular content of copper. Native gel shift experiments with the periplasmic N-terminal domain of the YcnJ membrane protein (135 residues) disclosed its strong affinity to Cu(II) ions in vitro. Inspection of the upstream sequence of
ycnJ
revealed that the
ycnK
gene encodes a putative transcriptional regulator, whose deletion caused an elevated expression of
ycnJ
, especially under conditions of copper excess. Further studies demonstrated that the recently identified copper efflux regulator CsoR also is involved in the regulation of
ycnJ
expression, leading to a new model for copper homeostasis in
B. subtilis
.
A Novel Integrative Conjugative Element Mediates Genetic Transfer from Group G Streptococcus to Other β-Hemolytic Streptococci
Authors: Davies Mark R. , Shera Josephine , Van Domselaar Gary H. , Sriprakash Kadaba S. , McMillan David J.
Published: January 2009

Lateral gene transfer is a significant contributor to the ongoing evolution of many bacterial pathogens, including β-hemolytic streptococci. Here we provide the first characterization of a novel integrative conjugative element (ICE), ICESde3396, fromStreptococcus dysgalactiaesubsp.equisimilis(group G streptococcus [GGS]), a bacterium commonly found in the throat and skin of humans. ICESde3396 is 64 kb in size and encodes 66 putative open reading frames. ICESde3396 shares 38 open reading frames with a putative ICE fromStreptococcus agalactiae(group B streptococcus [GBS]), ICESa2603. In addition to genes involves in conjugal processes, ICESde3396 also carries genes predicted to be involved in virulence and resistance to various metals. A major feature of ICESde3396 differentiating it from ICESa2603 is the presence of an 18-kb internal recombinogenic region containing four unique gene clusters, which appear to have been acquired from streptococcal and nonstreptococcal bacterial species. The four clusters include two cadmium resistance operons, an arsenic resistance operon, and genes with orthologues in a group A streptococcus (GAS) prophage. Streptococci that naturally harbor ICESde3396 have increased resistance to cadmium and arsenate, indicating the functionality of genes present in the 18-kb recombinogenic region. By marking ICESde3396 with a kanamycin resistance gene, we demonstrate that the ICE is transferable to other GGS isolates as well as GBS and GAS. To investigate the presence of the ICE in clinical streptococcal isolates, we screened 69 isolates (30 GGS, 19 GBS, and 20 GAS isolates) for the presence of three separate regions of ICESde3396. Eleven isolates possessed all three regions, suggesting they harbored ICESde3396-like elements. Another four isolates possessed ICESa2603-like elements. We propose that ICESde3396 is a mobile genetic element that is capable of acquiring DNA from multiple bacterial sources and is a vehicle for dissemination of this DNA through the wider β-hemolytic streptococcal population.
Transcriptional Regulation of the Cellobiose Operon of
Streptococcus mutans
Authors: Zeng Lin , Burne Robert A.
Published: January 2009

The ability of
Streptococcus mutans
to catabolize cellobiose, a β-linked glucoside generated during the hydrolysis of cellulose, is shown to be regulated by a transcriptional regulator, CelR, which is encoded by an operon with a phospho-β-glucosidase (CelA) and a cellobiose-specific sugar
p
hospho
t
ransferase
s
ystem (PTS) permease (EII
Cel
). The roles of CelR, EII
Cel
components, and certain fructose/mannose-PTS permeases in the transcriptional regulation of the
cel
locus were analyzed. The results revealed that (i) the
celA
and
celB
(EIIB
Cel
) gene promoters require CelR for transcriptional activation in response to cellobiose, but read-through from the
celA
promoter contributes to expression of the EII
Cel
genes; (ii) the EII
Cel
subunits were required for growth on cellobiose and for transcriptional activation of the
cel
genes; (iii) CcpA plays little direct role in catabolite repression of the
cel
regulon, but loss of specific PTS permeases alleviated repression of
cel
genes in the presence of preferred carbohydrates; and (iv) glucose could induce transcription of the
cel
regulon when transported by EII
Cel
. CelR derivatives containing amino acid substitutions for five conserved histidine residues in two PTS regulatory domains and an EIIA-like domain also provided important insights regarding the function of this regulator. Based on these data, a model for the involvement of PTS permeases and the general PTS proteins enzyme I and HPr was developed that reveals a critical role for the PTS in CcpA-independent catabolite repression and induction of
cel
gene expression in
S. mutans
.
An Intact Urease Assembly Pathway Is Required To Compete with NikR for Nickel Ions in
Helicobacter pylori
Authors: Benanti Erin L. , Chivers Peter T.
Published: January 2009

We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in
H. pylori
strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni
2+
-limiting conditions, as measured by reduced transcript levels and
63
Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.
The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence inEnterococcus faecalis
Authors: Abranches Jacqueline , Martinez Alaina R. , Kajfasz Jessica K. , Chávez Violeta , Garsin Danielle A. , Lemos José A.
Published: January 2009

The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production inEnterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of theE. faecalisgenome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of ΔrelA, ΔrelQ, and ΔrelAQstrains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the ΔrelAstrain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a ΔrelAQdouble mutant restored many of the stress-sensitive phenotypes of ΔrelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the ΔrelAstrain but diminished in the ΔrelQand ΔrelAQstrains. Finally, virulence of the ΔrelAQstrain but not of the ΔrelAor ΔrelQstrain was significantly attenuated in theCaenorhabditis elegansmodel. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.
Impact of parental home smoking policies on policy choices of independently living young adults
Authors: Albers A B , Biener L , Siegel M , Cheng D M , Rigotti N A
Published: January 2009

Objective:

To determine whether adolescents living in parental homes where smoking is banned are more likely to move into smoke-free living quarters when they leave home.

Methods:

We analysed data on 693 youths from a 4-year, three-wave prospective study of a representative sample of Massachusetts adolescents (aged 12–17). All youths resided in independent living quarters at follow-up. The primary outcome was presence of a smoking ban in the living quarters at follow-up. The primary predictor was presence of a household smoking ban in the parental home, assessed 2 years before the outcome. Generalised linear mixed effects models examined the effect of a parental household smoking ban on the odds of moving into smoke-free living quarters at follow-up overall and stratified by smoking status at follow-up.

Results:

Youths leaving home had much higher odds of moving to smoke-free living quarters if their parental household had had a smoking ban (odds ratio (OR) = 12.70, 95% CI, 6.19 to 26.04). Other independent predictors included moving into a school or college residence (OR = 3.88, 95% CI 1.87 to 8.05), and not living with smokers at follow-up (OR = 3.91, 95% CI 1.93 to 7.92).

Conclusions:

A household smoking ban in the parental home appears to lead youths to prefer smoke-free living quarters once they leave home.