bluuebunny/crossref_metadata_2025 huggingface dataset:
Serial Analysis of rRNA Genes and the Unexpected Dominance of Rare Members of Microbial Communities
Authors: Ashby Matthew N. , Rine Jasper , Mongodin Emmanuel F. , Nelson Karen E. , Dimster-Denk Dago
Published: May 2007

The accurate description of a microbial community is an important first step in understanding the roles of its components in ecosystem function. A method for surveying microbial communities termed serial analysis of rRNA genes (SARD) is described here. Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA genes from microbial communities. These tags are ligated to form concatemers comprised of 20 to 40 tags which are cloned and identified by DNA sequencing. Four agricultural soil samples were profiled with SARD to assess the method's utility. A total of 37,008 SARD tags comprising 3,127 unique sequences were identified. A comparison of duplicate profiles from one soil genomic DNA preparation revealed that the method was highly reproducible. The large numbers of singleton tags, together with nonparametric richness estimates, indicated that a significant amount of sequence tag diversity remained undetected with this level of sampling. The abundance classes of the observed tags were scale-free and conformed to a power law distribution. Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community. Over 99% of the unique tags individually made up less than 1% of the community. Therefore, from either a numerical or diversity standpoint, taxa with low abundance comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function. SARD may provide a means to explore the ecological roles of these rare members of microbial communities in qualitative and quantitative terms.
Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of
Mycobacterium ulcerans
in Clinical and Environmental Samples
Authors: Fyfe Janet A. M. , Lavender Caroline J. , Johnson Paul D. R. , Globan Maria , Sievers Aina , Azuolas Joseph , Stinear Timothy P.
Published: May 2007

Mycobacterium ulcerans
is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of
M. ulcerans
infection in humans and has been used for the detection of
M. ulcerans
in the environment. This paper describes the development of a TaqMan assay targeting IS
2404
multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of
M. ulcerans
infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS
2606
and the ketoreductase-B domain of the
M. ulcerans
mycolactone polyketide synthase genes was designed to augment the specificity of the IS
2404
PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of
M. ulcerans
DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.
New
Trypanosoma cruzi
Repeated Element That Shows Site Specificity for Insertion
Authors: Souza Renata T. , Santos Márcia R. M. , Lima Fábio M. , El-Sayed Najib M. , Myler Peter J. , Ruiz Jeronimo C. , da Silveira José Franco
Published: May 2007

A new family of site-specific repeated elements identified in
Trypanosoma cruzi
, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other
T. cruzi
sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5′ end of the element and a sequence domain of ∼500 bp without a well-defined borderline at the 3′ end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of ∼0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be ∼173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site.
Saccharomyces cerevisiae
Vacuole in Zinc Storage and Intracellular Zinc Distribution
Authors: Simm Claudia , Lahner Brett , Salt David , LeFurgey Ann , Ingram Peter , Yandell Brian , Eide David J.
Published: May 2007

Previous studies of the yeast
Saccharomyces cerevisiae
indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these methods, we determined that zinc levels in the vacuole vary with zinc status and can rise to almost 100 mM zinc (i.e., 7 × 10
8
atoms of vacuolar zinc per cell). Moreover, this zinc can be mobilized effectively to supply the needs of as many as eight generations of progeny cells under zinc starvation conditions. While the Zrc1 and Cot1 zinc transporters are essential for zinc uptake into the vacuole under steady-state growth conditions, additional transporters help mediate zinc uptake into the vacuole during “zinc shock,” when zinc-limited cells are resupplied with zinc. In addition, we found that other compartments of the cell do not provide significant stores of zinc. In particular, zinc accumulation in mitochondria is low and is homeostatically regulated independently of vacuolar zinc storage. Finally, we observed a strong correlation between zinc status and the levels of magnesium and phosphorus accumulated in cells. Our results implicate zinc as a major determinant of the ability of the cell to store these other important nutrients.
Protective Immune Responses to a Recombinant Adenovirus Type 35 Tuberculosis Vaccine in Two Mouse Strains: CD4 and CD8 T-Cell Epitope Mapping and Role of Gamma Interferon
Authors: Radošević Katarina , Wieland Catharina W. , Rodriguez Ariane , Weverling Gerrit Jan , Mintardjo Ratna , Gillissen Gert , Vogels Ronald , Skeiky Yasir A. W. , Hone David M. , Sadoff Jerald C. , van der Poll Tom , Havenga Menzo , Goudsmit Jaap
Published: May 2007

There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of
Mycobacterium tuberculosis
antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against
M. tuberculosis
when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-γ)-producing T cells can provide protection against
M. tuberculosis
challenge. While in BALB/c (H-2
d
) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2
b
) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-γ.
Transcriptome Analysis of Murine Macrophages in Response to Infection withStreptococcus pyogenesReveals an Unusual Activation Program
Authors: Goldmann Oliver , von Köckritz-Blickwede Maren , Höltje Claudia , Chhatwal Gursharan S. , Geffers Robert , Medina Eva
Published: May 2007

The complex response of murine macrophages to infection withStreptococcus pyogeneswas investigated at the level of gene expression with a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encode molecules involved in the immune response and in inflammation, transcription, signaling, apoptosis, the cell cycle, electron transport, and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype), such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, and as well as the up-regulation of anti-inflammatory mediators, such as IL-1 decoy receptor and IL-10, associated with alternative macrophage activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation, was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine involved in the alternative activation pathway, was up-regulated inS. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated thatS. pyogenesinduces an atypical activation program in macrophages, with some but not all features of the classical or alternative activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages againstS. pyogenesis mediated by phagocyte oxidase, asp47phoxwas up-regulated in infected cells. Indeed, the in vivo and in vitro killing ofS. pyogeneswas markedly diminished in the absence of functional phagocyte (p47phox−/−) but not in the absence of iNOS (iNOS−/−). An understanding of how macrophages respond toS. pyogenesat the molecular level may facilitate the development of new therapeutic paradigms.
Nitrate Sensing and Metabolism Modulate Motility, Biofilm Formation, and Virulence in
Pseudomonas aeruginosa
Authors: Van Alst Nadine E. , Picardo Kristin F. , Iglewski Barbara H. , Haidaris Constantine G.
Published: May 2007

Infection by the bacterial opportunist
Pseudomonas aeruginosa
frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by
P. aeruginosa
is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type
P. aeruginosa
PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in
P. aeruginosa
motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for
P. aeruginosa
pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host
Caenorhabditis elegans
. Interestingly, the membrane nitrate reductase mutant was avirulent in
C. elegans
, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in
P. aeruginosa
pathogenesis.
Adherence-Inhibitory Intestinal Immunoglobulin A Antibody Response in Baboons Elicited by Use of a Synthetic Intranasal Lectin-Based Amebiasis Subunit Vaccine
Authors: Abd Alla Mohamed D. , White Gary L. , Rogers Tyson B. , Cary Max E. , Carey David W. , Ravdin Jonathan I.
Published: May 2007

We designed an amebiasis subunit vaccine that is constructed by using four peptide epitopes of the galactose-inhibitable lectin heavy subunit that were recognized by intestinal secretory immunoglobulin A (IgA) antibodies from immune human subjects. These epitopes are contained in the region encompassing amino acids 758 to 1134 of the lectin heavy subunit, designated LC3. Baboons (Papio anubis) are natural hosts forEntamoeba histolytica; naturally infected baboons raised in captivity possess serum IgA antibodies to the same four LC3 epitopes as humans. Uninfected, seronegative baboons received four intranasal immunizations at 7-day intervals with the synthetic peptide vaccine (400, 800, or 1,600 μg per nostril) with cholera toxin (20 μg) as the adjuvant. As determined by an enzyme-linked immunosorbent assay (ELISA), each dose of the peptide vaccine elicited antipeptide serum IgA and IgG and intestinal IgA antibody responses in all six immunized baboons by day 28, 7 days after the last immunization (P, <0.01 for each dose compared to the cholera toxin control). The peptide vaccine elicited serum IgG and intestinal IgA antibodies that recognized purified recombinant LC3 protein (P, <0.008 and 0.02, respectively) and native lectin protein (P< 0.01). In addition, an indirect immunofluorescence assay with whole trophozoites (P< 0.01) and Western blot analysis confirmed that serum IgG antibodies from vaccinated baboons recognized native lectin protein on the surfaces of axenicE. histolyticatrophozoites or from solubilized amebae. All four synthetic peptides were immunogenic; the vaccine elicited dose- and time-dependent responses, as determined by ELISA optical density readings indicating the production of serum and intestinal antibodies (P, <0.02 for antipeptide and antilectin antibodies). As a positive control, intranasal immunization with purified recombinant LC3 protein with cholera toxin as the adjuvant elicited a serum anti-LC3 IgA and IgG antibody response (P, 0.05 and <0.0001, respectively); however, no intestinal anti-LC3 IgA antibody response was observed (P= 0.4). Of interest, serum IgA and IgG antibodies elicited by the recombinant LC3 vaccine did not recognize any of the four putatively protective LC3 peptide epitopes. Both serum and fecal antibodies elicited by the peptide vaccine exhibited neutralizing activity, as determined by their dose-dependent inhibition of the galactose-specific adherence ofE. histolyticatrophozoites to Chinese hamster ovary cells in vitro (P, <0.001 for each group of antibodies compared to the control). In summary, a lectin-based intranasal polylysine-linked synthetic peptide vaccine was effective in eliciting an adherence-inhibitory, intestinal antilectin IgA antibody response in baboons. Future studies with the baboon model will determine vaccine efficacy against asymptomaticE. histolyticaintestinal infection.
Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils
Authors: Herrmann Jens Martin , Bernardo John , Long Heidi J. , Seetoo Kurt , McMenamin Mary E. , Batista Eraldo L. , Van Dyke Thomas E. , Simons Elizabeth R.
Published: May 2007

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants’ origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Δ[Ca2+]iand ΔpHi) and of bactericidal entities (oxidative species and elastase) exposed toN-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca2+]i, mostly from intracellular stores, simultaneously with a drop in pHi; these are followed by a drop in [Ca2+]iand a rise in pHi, with the latter being due to a Na+/H+antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca2+]iis restored, 10−7M fMLP, previously shown to elicit maximal Δ[Ca2+]ibut no bactericidal functions, did not prevent the cells’ responses with Δ[Ca2+]ito a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 μg/ml IC, previously shown to elicit maximal Δ[Ca2+]iand bactericidal functions, exhibited no subsequent Δ[Ca2+]ior ΔpHito either stimulus. While exposure to 10−7M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10−5M fMLP did, presumably via the low-affinity receptor, using a different Ca2+source.
Induction of Nitric Oxide Synthase and Activation of Signaling Proteins inAnophelesMosquitoes by the Malaria Pigment, Hemozoin
Authors: Akman-Anderson Leyla , Olivier Martin , Luckhart Shirley
Published: May 2007

Anopheles stephensi, a major vector for malaria parasite transmission, responds toPlasmodiuminfection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown thatPlasmodium falciparumglycosylphosphatidylinositols (PfGPIs) can induceA. stephensiNO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In mosquito cells, signaling by PfGPIs andP. falciparummerozoites is mediated through Akt/protein kinase B (Akt/PKB), the mitogen-activated protein kinase kinase DSOR1, and extracellular signal-regulated kinase (ERK). In mammalian cells, a second parasite factor, malaria pigment or hemozoin (Hz), signals NOS induction through ERK- and nuclear factor kappa B-dependent pathways and has been demonstrated to be a novel proinflammatory ligand for Toll-like receptor 9. In this study, we demonstrate that Hz can also induce AsNOS gene expression in immortalizedA. stephensiandAnopheles gambiaecell lines in vitro and inA. stephensimidgut tissue in vivo. In mosquito cells, Hz signaling is mediated through transforming growth factor β-associated kinase 1, Akt/PKB, ERK, and atypical protein kinase C zeta/lambda. Our results show that Hz is a prominent parasite-derived signal forAnophelesand that signaling pathways activated by PfGPIs and Hz have both unique and shared components. Together with our previous findings, our data indicate that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.
Soluble CD14 and CD83 from Human Neonatal Antigen-Presenting Cells Are Inducible by Commensal Bacteria and Suppress Allergen-Induced Human Neonatal Th2 Differentiation
Authors: Lundell Anna-Carin , Andersson Kerstin , Josefsson Elisabet , Steinkasserer Alexander , Rudin Anna
Published: May 2007

CD14 is expressed on the cell surface of various antigen-presenting cells, and CD83 is a maturation marker for dendritic cells (DC). CD14 and CD83 are also present as soluble proteins, and both have immunoregulatory functions. We examined whether neonatal cord blood monocytes or DC released soluble CD14 (sCD14) or sCD83 when exposed to the commensal intestinal bacteriaClostridium perfringens,Staphylococcus aureus,Lactobacillus rhamnosus,Escherichia coli, andBacteroides fragilis. We found that the gram-positive bacteriaC. perfringensandS. aureus, but not gram-negative bacteria, induced the release of sCD14 from monocytes. DC, on the other hand, released sCD14 in response to both gram-positive and gram-negative bacteria. Moreover, the expression of the virulence factor staphylococcal protein A seemed to be important forS. aureus-induced sCD14 production from both monocytes and DC. Soluble CD83 was released from DC, but not from monocytes, when exposed to both gram-positive and gram-negative bacteria. Finally, to investigate whether sCD14 or sCD83 could modulate neonatal allergen-induced T-cell differentiation, DC were exposed to birch allergen alone or in the presence of sCD14 or sCD83 and then cocultured with autologous T cells. We demonstrate that sCD14 and sCD83 inhibited the birch allergen-induced Th2 differentiation by suppressing interleukin 13 production. Together, these results suggest that the commensal intestinal flora may be an important stimulus for the developing immune system by inducing the immunoregulatory proteins sCD14 and sCD83, which may be involved in preventing T-cell sensitization to allergens in infants.
Interactions between the Lipoprotein PilP and the Secretin PilQ inNeisseria meningitidis
Authors: Balasingham Seetha V. , Collins Richard F. , Assalkhou Reza , Homberset Håvard , Frye Stephan A. , Derrick Jeremy P. , Tønjum Tone
Published: May 2007

Neisseria meningitidiscan be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, sincepilPmutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP intrans. ThepilPgene is located upstream ofpilQ, and analysis of their transcripts indicated thatpilPandpilQwere cotranscribed. Furthermore, analysis of the level of PilQ expression inpilPmutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect onpilQtranscription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions.
Gene Expression Analysis of
Corynebacterium glutamicum
Subjected to Long-Term Lactic Acid Adaptation
Authors: Jakob Kinga , Satorhelyi Peter , Lange Christian , Wendisch Volker F. , Silakowski Barbara , Scherer Siegfried , Neuhaus Klaus
Published: May 2007

Corynebacteria form an important part of the red smear cheese microbial surface consortium. To gain a better understanding of molecular adaptation due to low pH induced by lactose fermentation, the global gene expression profile of
Corynebacterium glutamicum
adapted to pH 5.7 with lactic acid under continuous growth in a chemostat was characterized by DNA microarray analysis. Expression of a total of 116 genes was increased and that of 90 genes was decreased compared to pH 7.5 without lactic acid, representing 7% of the genes in the genome. The up-regulated genes encode mainly transcriptional regulators, proteins responsible for export, import, and metabolism, and several proteins of unknown function. As much as 45% of the up-regulated open reading frames code for hypothetical proteins. These results were validated using real-time reverse transcription-PCR. To characterize the functions of 38 up-regulated genes, 36 single-crossover disruption mutants were generated and analyzed for their lactic acid sensitivities. However, only a
sigB
knockout mutant showed a highly significant negative effect on growth at low pH, suggesting a function in organic-acid adaptation. A
sigE
mutant already displayed growth retardation at neutral pH but grew better at acidic pH than the
sigB
mutant. The lack of acid-sensitive phenotypes in 34 out of 36 disrupted genes suggests either a considerable redundancy in acid adaptation response or coincidental effects. Other up-regulated genes included genes for ion transporters and metabolic pathways, including carbohydrate and respiratory metabolism. The enhanced expression of the
nrd
(ribonucleotide reductase) operon and a DNA ATPase repair protein implies a cellular response to combat acid-induced DNA damage. Surprisingly, multiple iron uptake systems (totaling 15% of the genes induced ≥2-fold) were induced at low pH. This induction was shown to be coincidental and could be attributed to iron-sequestering effects in complex media at low pH.
The Two-Component Regulatory System
senX3-regX3
Regulates Phosphate-Dependent Gene Expression in
Mycobacterium smegmatis
Authors: Glover Robert T. , Kriakov Jordan , Garforth Scott J. , Baughn Anthony D. , Jacobs William R.
Published: May 2007

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P
i
) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P
i
acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the
senX3-regX3
2CR as a P
i
-dependent regulator of genes involved in phosphate acquisition in
Mycobacterium smegmatis
. Characterization of
senX3
mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon P
i
availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3∼P) as a transcriptional activator of
phoA
. Furthermore, purified RegX3∼P bound to promoter sequences from
phoA
,
senX3
, and the high-affinity phosphate transporter component
pstS
, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the
phoA
promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3∼P. Based upon our findings, we propose a model for P
i
-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.
Functional Incorporation of Chimeric
b
Subunits into F
1
F
o
ATP Synthase
Authors: Claggett Shane B. , Grabar Tammy Bohannon , Dunn Stanley D. , Cain Brian D.
Published: May 2007

F
1
F
o
ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F
1
sector and its catalytic sites against the movement of the rotor. In
Escherichia coli
, the peripheral stalk is a homodimer of identical
b
subunits, but photosynthetic bacteria have open reading frames for two different
b
-like subunits thought to form heterodimeric
b
/
b
′ peripheral stalks. Chimeric
b
subunit genes have been constructed by substituting sequence from the
Thermosynechococcus elongatus b
and
b
′ genes in the
E. coli uncF
gene, encoding the
b
subunit. The recombinant genes were expressed alone and in combination in the
E. coli
deletion strain KM2 (Δ
b
). Although not all of the chimeric subunits were incorporated into F
1
F
o
ATP synthase complexes, plasmids expressing either chimeric
b
E39-I86
or
b
′
E39-I86
were capable of functionally complementing strain KM2 (Δ
b
). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the
b
′
E39-D53
or
b
L54-I86
subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the
T. elongatus b
subunit proved to be more stable than the
b
′ subunit in vitro. Coexpression of the
b
E39-I86
and
b
′
E39-I86
subunits in strain KM2 (Δ
b
) yielded F
1
F
o
complexes containing heterodimeric peripheral stalks composed of both subunits.
The Pneumococcal Two-Component Signal Transduction System RR/HK06 Regulates CbpA and PspA by Two Distinct Mechanisms
Authors: Standish Alistair J. , Stroeher Uwe H. , Paton James C.
Published: May 2007

We have previously shown that CbpA, a major pneumococcal virulence factor, is regulated by the two-component signal transduction system RR/HK06 (A. J. Standish, U. H. Stroeher, and J. C. Paton, Proc. Natl. Acad. Sci. USA 102:7701-7706, 2005). However, additional unidentified regulated factors appeared to be responsible for differences in adherence and the ability ofStreptococcus pneumoniaeto cause disease in a mouse model. Here, we identified a number of other regulated genes by overexpressing the system.cbpA, along with a cotranscribed upstream gene, showed substantial increases in expression when RR06 was overexpressed inS. pneumoniaestrains D39 and TIGR4. However, there were no other similarities between these strains. In D39,rr06overexpression decreased expression of numerous factors, including the major virulence factor genepspA. Further investigation ofcbpAregulation by RR/HK06, using mutants with mutations in both HK06 and RR06, suggested that rather than the norm,cbpAtranscription was activated when RR06 was in the nonphosphorylated form. Although other factors, such aspspAandgls24, are regulated by this system, these genes appear to be repressed when RR06 is in its phosphorylated form.
Characterization of the Fucosylation Pathway in the Biosynthesis of Glycopeptidolipids fromMycobacterium aviumComplex
Authors: Miyamoto Yuji , Mukai Tetsu , Maeda Yumi , Nakata Noboru , Kai Masanori , Naka Takashi , Yano Ikuya , Makino Masahiko
Published: May 2007

The cell envelopes of several species of nontuberculous mycobacteria, including theMycobacterium aviumcomplex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of theM. aviumcomplex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of theM. aviumcomplex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinantMycobacterium smegmatisstrain that possesses thertfAgene of theM. aviumcomplex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genesmdhtA,merA, andgtfDare indispensable for the fucosylation. Functional characterization revealed that thegtfDgene encodes a glycosyltransferase that transfers a fucose residue via 1→3 linkage to a rhamnose residue of serovar 1 GPL. The other two genes,mdhtAandmerA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of themdhtA,merA, andgtfDgenes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from theM. aviumcomplex, which are structurally distinct.
Functional Characterization of the
Sinorhizobium meliloti
Acetate Metabolism Genes
aceA
, SMc00767, and
glcB
Authors: Ramírez-Trujillo J. A. , Encarnación S. , Salazar E. , de los Santos A. García , Dunn M. F. , Emerich D. W. , Calva E. , Hernández-Lucas I.
Published: May 2007

The genes encoding malate synthase (
glcB
) and isocitrate lyase (
aceA
) and a 240-bp open reading frame (SMc00767) located downstream of
aceA
were isolated and functionally characterized in
Sinorhizobium meliloti
. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed.
aceA
mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in
glcB
grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that
aceA
and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants.
aceA
-
glcB
,
aceA
-SMc00767, and
glcB
-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type
aceA
-
glcB
or
aceA
-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of
aceA
and SMc00767 and show that
glcB
, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains.
aceA
and
glcB
were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates
aceA
expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including
aceA
; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in
S. meliloti
. Genetic and functional analyses indicated that
aceA
and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with the
aceA
and
glcB
mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the
Rhizobium
-legume symbiosis.
Differential Interaction ofAggregatibacter(Actinobacillus)actinomycetemcomitansLsrB and RbsB Proteins with Autoinducer 2
Authors: Shao Hanjuan , James Deanna , Lamont Richard J. , Demuth Donald R.
Published: May 2007

Our previous studies showed that theAggregatibacter actinomycetemcomitansRbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that therbsDABCKoperon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However,A. actinomycetemcomitansalso possesses genes related to thelsroperon ofSalmonella entericaserovar Typhimurium which function to import AI-2. Here, we show thatA. actinomycetemcomitansLsrB protein competitively inhibits the interaction of theVibrio harveyiAI-2 receptor (LuxP) with AI-2 from eitherA. actinomycetemcomitansorV. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 fromV. harveyiwhereas RbsB competed more effectively with LuxP forA. actinomycetemcomitansAI-2. Inactivation oflsrBin wild-typeA. actinomycetemcomitansor in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depletedA. actinomycetemcomitansAI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of bothlsrBandrbsBvirtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest thatA. actinomycetemcomitanspossesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.
The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome
Authors: Alcorlo Martín , González-Huici Víctor , Hermoso José M. , Meijer Wilfried J. J. , Salas Margarita
Published: May 2007

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of
Bacillus subtilis
bacteriophage φ29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step ∼65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.
Inactivation of the DnaB Helicase Leads to the Collapse and Degradation of the Replication Fork: a Comparison to UV-Induced Arrest
Authors: Belle Jerilyn J. , Casey Andrew , Courcelle Charmain T. , Courcelle Justin
Published: May 2007

Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including
recF
,
recO
,
recR
,
recJ
,
recG
, and
xonA
. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.
Double-Strand Break Generation under Deoxyribonucleotide Starvation in
Escherichia coli
Authors: Guarino Estrella , Salguero Israel , Jiménez-Sánchez Alfonso , Guzmán Elena C.
Published: May 2007

Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo “replication fork reversal.” This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.
Identification ofPseudomonas syringaepv. syringae 61 Type III Secretion System Hrp Proteins That Can Travel the Type III Pathway and Contribute to the Translocation of Effector Proteins into Plant Cells
Authors: Ramos Adela R. , Morello Joanne E. , Ravindran Sandeep , Deng Wen-Ling , Huang Hsiou-Chen , Collmer Alan
Published: May 2007

Pseudomonas syringaetranslocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.
FepA- and TonB-Dependent Bacteriophage H8: Receptor Binding and Genomic Sequence
Authors: Rabsch Wolfgang , Ma Li , Wiley Graham , Najar Fares Z. , Kaserer Wallace , Schuerch Daniel W. , Klebba Joseph E. , Roe Bruce A. , Gomez Jenny A. Laverde , Schallmey Marcus , Newton Salete M. C. , Klebba Phillip E.
Published: May 2007

H8 is derived from a collection of
Salmonella enterica
serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family
Siphoviridae
. H8 infected
S. enterica
serotypes Enteritidis and Typhimurium and
Escherichia coli
by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to
E. coli
FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but
exbB
mutations in
Salmonella
or
E. coli
did not prevent infection; only
exbB tolQ
or
exbB tolR
double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process.
An Anhydro-
N
-Acetylmuramyl-
l
-Alanine Amidase with Broad Specificity Tethered to the Outer Membrane of
Escherichia coli
Authors: Uehara Tsuyoshi , Park James T.
Published: May 2007

From its amino acid sequence homology with AmpD, we recognized YbjR, now renamed AmiD, as a possible second 1,6-anhydro-
N
-acetylmuramic acid (anhMurNAc)-
l
-alanine amidase in
Escherichia coli
. We have now confirmed that AmiD is an anhMurNAc-
l
-Ala amidase and demonstrated that AmpD and AmiD are the only enzymes present in
E. coli
that are able to cleave the anhMurNAc-
l
-Ala bond. The activity was present only in the outer membrane fraction obtained from an
ampD
mutant. In contrast to AmpD, which is specific for the anhMurNAc-
l
-alanine bond, AmiD also cleaved the bond between MurNAc and
l
-alanine in both muropeptides and murein sacculi. Unlike the periplasmic murein amidases, AmiD did not participate in cell separation.
ampG
mutants, which are unable to import GlcNAc-anhMurNAc-peptides into the cytoplasm, released mainly peptides into the medium due to AmiD activity, whereas an
ampG amiD
double mutant released a large amount of intact GlcNAc-anhMurNAc-peptides into the medium.
Bifunctional CTP:Inositol-1-Phosphate Cytidylyltransferase/CDP-Inositol:Inositol-1-Phosphate Transferase, the Key Enzyme for Di-myo-Inositol-Phosphate Synthesis in Several (Hyper)thermophiles
Authors: Rodrigues Marta V. , Borges Nuno , Henriques Mafalda , Lamosa Pedro , Ventura Rita , Fernandes Chantal , Empadinhas Nuno , Maycock Christopher , da Costa Milton S. , Santos Helena
Published: May 2007

The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts ofArchaeoglobus fulgidusand structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol.188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression inEscherichia coliof the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3′-phosphate-1′-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus,Pyrococcus furiosus,Thermococcus kodakaraensis,Aquifex aeolicus, andRubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP andl-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol andl-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP andl-myo-inositol-1-P; the DIPP synthase domain used onlyl-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted inAeropyrum pernix,Thermotoga maritima, andHyperthermus butylicus. Additionally, usingl-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3′-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.