bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulins in Nasal Secretions of Healthy Humans: Structural Integrity of Secretory Immunoglobulin A1 (IgA1) and Occurrence of Neutralizing Antibodies to IgA1 Proteases of Nasal Bacteria
Authors: Kirkeby Line , Rasmussen Trine Tang , Reinholdt Jesper , Kilian Mogens
Published: April 2009

Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) μg ml−1. IgA1 protease-producing bacteria (Haemophilus influenzae,Streptococcus pneumoniae, orStreptococcus mitisbiovar 1) were isolated from the nasal cavities of seven subjects at 2.1 × 103to 7.2 × 106CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, α-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous isolates were detected in secretions from five of the seven subjects but not in those from two subjects harboring IgA1 protease-producingS. mitisbiovar 1. α-chain fragments different from Fcαand Fdαwere detected in some samples, possibly reflecting nonspecific proteolytic activity of microbial or host origin. These results add to previous evidence for a role of secretory immunity in the defense of the nasal mucosa but do not help identify conditions under which bacterial IgA1 proteases may interfere with this defense.
Antibody Responses to Antigenic Sites 1 and 3 of Serotype 3 Poliovirus after Vaccination with Oral Live Attenuated or Inactivated Poliovirus Vaccine and after Natural Exposure
Authors: Herremans T. , Reimerink J. H. J. , Kimman T. G. , van der Avoort H. G. A. M. , Koopmans M. P. G.
Published: April 2009

Three important antigenic sites involved in virus neutralization on polioviruses in mouse experiments have been identified. These sites are located at the surface of the virion and have been designated antigenic sites 1, 2, and 3. In mice, the antibody response to antigenic site 1 of serotype 3 poliovirus is considered to be immunodominant, but little is known about the immunogenicity of these sites in humans. In the present study, we developed inhibition enzyme-linked immunosorbent assays specific for antigenic sites 1 and 3 to measure antibody responses to these sites in fully vaccinated inactivated poliovirus vaccine (IPV) (n= 63) and oral live attenuated poliovirus vaccine (OPV) (n= 63) recipients and in naturally infected persons (n= 25). Similar levels of antibodies to site 1 in IPV and OPV vaccinees were detected. However, significantly more OPV recipients (88.7%) had detectable antibodies to antigenic site 3 (P< 0.01) than did IPV-vaccinated persons (63.1%). After an IPV booster vaccination, both previously IPV- and OPV-vaccinated persons responded with a significant increase in antibodies to sites 1 and 3 (P< 0.01). We conclude that the immune response to serotype 3 poliovirus in humans consists of both site 1- and site 3-specific antibodies and that these responses can be induced by either OPV or recent IPV vaccination.
Optimization of the Weck-Cel Collection Method for Quantitation of Cytokines in Mucosal Secretions
Authors: Rohan Lisa Cencia , Edwards Robert P. , Kelly Lori A. , Colenello Kelly A. , Bowman Frederick P. , Crowley-Nowick Peggy A.
Published: April 2009

Measurement of immune components in mucosal secretions is important for the evaluation of local immunity at the mucosal surfaces. The Weck-Cel ophthalmic sponge provides a method for the collection of these secretions. The sponge absorbs a relatively large volume of material, therefore allowing for quantitation of multiple immune components. Additionally, it provides a method in which the same device may be used to collect specimens from different mucosal sites, such as the genital tract and oral cavity. This sampling technique has successfully been applied for collection and measurement of antibody in oral and genital tract secretions. The purpose of this work was to optimize the extraction of protein from the sponge matrix. Of particular interest was the recovery of cytokines from the sponge. Satisfactory recovery of the cytokines interleukin 1β (IL-1β), IL-2, IL-5, IL-12, IL-6, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor was obtained. However, IL-4 and gamma interferon recovery rates remained low. Using an alteration of the published extraction method, cytokine concentrations were measured in cervical secretions from women using oral contraceptives. The data revealed detectable concentrations of IL-6, IL-10, IL-8, and IL-12 on cycle days 9 and 20. The proposed technique provides an easy, practical, and consistent method for collection of nonconventional body fluids, such as cervicovaginal fluids and saliva, for the assay of immunoglobulins and several cytokines.
An In Vitro Microbial-Caries Model Used to Study the Efficacy of Antibodies toStreptococcus mutansSurface Proteins in Preventing Dental Caries
Authors: Fontana Margherita , Buller Tiffany L. , Dunipace Ann J. , Stookey George K. , Gregory Richard L.
Published: April 2009

The first step for a pathogenic bacterium to initiate infection is via attachment (i.e., through surface determinants) to a suitable receptor. An in vitro microbial artificial-mouth model was used to test the efficacy of polyclonal antibodies toStreptococcus mutanscell surface proteins (CsAb) and a cell surface 59-kDa protein (59Ab) in preventingS. mutanscolonization and carious lesion formation. In study 1, groups of 12 human teeth specimens were inoculated withS. mutans, which were incubated with different concentrations of CsAb (A1 [positive control], sterile saline, no antibody; A2, 0.007 mg of antibody protein/ml; and A3, 0.7 mg of antibody protein/ml) for 1 h at 37°C. The negative control group (B1) was not infected and was incubated with Trypticase soy broth (TSB) without dextrose supplemented with 5% sucrose (TSBS). In study 2, the same study design was used except that 59Ab was used instead of CsAb, normal rabbit serum was used in the positive control group (A1), and TSB supplemented with 1% glucose was used as the nutrient to control for sucrose-dependent colonization. All groups were exposed for 4 days to circulating cycles of TSBS and TSB (study 1 and study 2, respectively; 30 min each, three times per day) and a mineral washing solution (21 h per day). Prior to each nutrient cycle, 1 ml of the appropriate CsAb or 59Ab solution was administered to each group and allowed to mix for 30 min before cycling was resumed. Data obtained by confocal laser scanning microscopy demonstrated the presence of a significantly smaller (P< 0.05) lesion area and a smaller total lesion fluorescence in group A3 than in group A1 for both studies. In study 1, group A2 had significantly smaller values than A1 for lesion depth and area. There were no significant differences between groups A2 and A3 for lesion area or between groups A1 and A2 for total lesion fluorescence. In study 2, there were no significant differences among groups A1 and A2 for lesion depth or between groups A2 and A3 for all of the parameters studied. In both studies, there were no significant differences betweenS. mutansplaque CFU numbers among any of the groups. These studies demonstrated the efficacy of CsAb and 59Ab in reducing primary caries development in this model, although the underlying mechanism remains unclear.
Diagnosis of Human Immunodeficiency Virus Infection Using an Immunoglobulin E-Based Assay
Authors: Fletcher MaryAnn , Miguez-Burbano Maria J. , Shor-Posner Gail , Lopez Viola , Lai Hong , Baum Marianna K.
Published: April 2009

Immunoglobulin assays that are sensitive and specific for detecting human immunodeficiency virus type 1 (HIV-1) infection are especially important in developing countries where PCR and viral culture may not be readily available. Immunoglobulin E (IgE), which is elevated in HIV-1 infection, is the only antibody that does not cross the placenta, making it potentially valuable for viral detection in both children and adults. This study developed an assay for detection of HIV specific IgE antibodies in adults. A total of 170 serum samples from 170 adults (116 HIV positive and 54 HIV negative) were analyzed. Serum or plasma samples were treated by using the protein G affinity method. The HIV status was determined by using two IgG enzyme-linked immunosorbent assays (ELISAs) and one Western blot evaluation. The IgE enzyme immunoassay test for HIV-1 correctly identified the HIV status in 98.8% of the samples (168 of 170). One false-positive and one false-negative test occurred with the IgE ELISA, as well as with the IgG ELISA test but were correctly identified by the IgE test. Analysis of the data demonstrated a high specificity (99%) and sensitivity (99%) of the IgE test, with 95% confidence intervals. The IgE assay appears to be sensitive and specific, suggesting that IgE-specific antibodies offer an effective method to detect HIV-1 infection in adults.
Validation of Immune-Complex Enzyme Immunoassays for Diagnosis of Pneumococcal Pneumonia among Adults in Kenya
Authors: Scott J. A. G. , Hall A. J. , Leinonen M.
Published: April 2009

The efficacy of pneumococcal vaccines in protecting against pneumococcal pneumonia can feasibly be measured only with a diagnostic technique that has a high specificity (0.98 to 1.00) and a sensitivity greatly exceeding that of blood cultures (>0.2 to 0.3). In this context immune-complex enzyme immunoassays (EIAs) offer a novel, convenient diagnostic method, and we have investigated three such assays with appropriate study populations in Kenya. Sera from 129 Kenyan adults with pneumococcal pneumonia and 97 ill controls from the same clinics, but without pneumococcal disease syndromes, were assayed with immune-complex EIAs for pneumolysin, C-polysaccharide, and mixed capsular polysaccharides (Pneumovax II). At an optical density (OD) threshold yielding a specificity of 0.95, the sensitivities (95% confidence intervals) of the assays were 0.22 (0.15 to 0.30), 0.26 (0.19 to 0.34), and 0.22 (0.15 to 0.29), respectively. For pneumolysin immune complexes, human immunodeficiency virus (HIV)-positive patients had a higher mean OD than HIV-negative patients (639 versus 321;
P
< 0.0001), but stratification by HIV infection status did not alter the performance of this test. Combining the results of all three EIAs did not enhance the diagnostic performances of the individual assays. In Kenyan adults the sensitivities of the immune-complex EIAs could exceed that of blood cultures only at levels of specificity that were insufficient for the performance of vaccine efficacy studies.
Human Granulocytic Ehrlichiosis Agent Infection in a Pony Vaccinated with aBorrelia burgdorferiRecombinant OspA Vaccine and Challenged by Exposure to Naturally Infected Ticks
Authors: Chang Yung-Fu , McDonough Sean P. , Chang Chao-Fu , Shin Kwang-Soon , Yen William , Divers Thomas
Published: April 2009

A pony was vaccinated with recombinant OspA vaccine (rOspA) and then exposed 3 months later toBorrelia burgdorferi-infected ticks (Ixodes scapularis) collected in Westchester County, N.Y. At 2 weeks after tick exposure, the pony developed a high fever (105°F). Buffy coat smears showed that 20% of neutrophils contained ehrlichial inclusion bodies (morulae). Flunixin Meglumine (1 g daily) was given for 2 days, and the body temperature returned to normal. PCR for ehrlichial DNA was performed on blood samples for 10 consecutive days beginning when the pony was first febrile. This pony was monitored for another 3.5 months but developed no further clinical signs. The 44-kDa immunodominant human granulocytic ehrlichiosis antigen gene was amplified by PCR and cloned into a pCR2.1 vector. DNA sequence analysis of this gene showed it was only 8 bp different (99% identity) from the results reported by others (J.W. Ijdo et al., Infect. Immun. 66:3264–3269, 1998). Western blot analysis, growth inhibition assays, and repeated attempts to isolateB. burgdorferiall demonstrated the pony was protected againstB. burgdorferiinfection. These results highlight the potential for ticks to harbor and transmit several pathogens simultaneously, which further complicates the diagnosis and vaccination of these emerging tick-borne diseases.
Opsonization and Phagocytosis ofPlasmodium falciparumMerozoites Measured by Flow Cytometry
Authors: Kumaratilake Lakshmi M. , Ferrante Antonio
Published: April 2009

A flow cytometric phagocytosis assay was established to investigate the role of anti-merozoite antibody, complement, and cytokines on the phagocytosis ofPlasmodium falciparummerozoites by human neutrophils. This assay involved allowing fluorescein isothiocyanate-labeled merozoites to interact with phagocytes and analysis of the cells on a FACScan with Lysis II software. To differentiate the proportion of neutrophil surface-bound merozoites from the merozoites ingested by neutrophils, the fluorescence of bound merozoites was quenched by adding trypan blue. The data showed that sera from malaria-immune individuals in the Solomon Islands and Papua New Guinea promoted merozoite engulfment by neutrophils. The cytokines tumor necrosis factor alpha, gamma interferon, granulocyte-macrophage colony-stimulating factor, and interleukin-1β significantly increased the amount and the rate of merozoite phagocytosis by neutrophils. Optimum merozoite phagocytosis occurred when both cytokines and anti-malarial antibody were present.
Interleukin-12 Is the Optimum Cytokine To Expand Human Th17 Cells In Vitro
Authors: Nady Soad , Ignatz-Hoover James , Shata Mohamed T.
Published: April 2009

Recently, a new lineage of CD4+T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+cells from the CD4+T cells. Functional studies revealed that CD4+IL-23R+cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-γ). Furthermore, we expanded the CD4+IL-23R+cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+IL-23R+cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-γ secreted by IL-23R+CD4+cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+CD4+cells and the secretion of IFN-γ, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.
Identification of Secreted Antigen 3 fromBabesia gibsoni
Authors: Jia Honglin , Terkawi M. Alaa , Aboge Gabriel Oluga , Goo Youn-Kyoung , Ma Liqing , Zhou Jinlin , Nishikawa Yoshifumi , Igarashi Ikuo , Fujisaki Kozo , Xuan Xuenan
Published: April 2009

A cDNA expression library ofBabesia gibsoniwas screened with the serum collected from a dog experimentally infected withB. gibsoni. A novel antigen sharing homology with secreted antigen 1 ofB. gibsoniwas isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome ofB. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed inEscherichia colicould recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected withB. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.
Relevance of Gamma Interferon, Tumor Necrosis Factor Alpha, and Interleukin-10 Gene Polymorphisms to Susceptibility to Mediterranean Spotted Fever
Authors: Forte Giusi Irma , Scola Letizia , Misiano Gabriella , Milano Salvatore , Mansueto Pasquale , Vitale Giustina , Bellanca Fiamma , Sanacore Maria , Vaccarino Loredana , Rini Giovan Battista , Caruso Calogero , Cillari Enrico , Lio Domenico , Mansueto Serafino
Published: April 2009

The acute phase of Mediterranean spotted fever (MSF) is characterized by dramatic changes in cytokine production patterns, clearly indicating their role in the immunomodulation of the response against the microorganism, and the differences in cytokine production seem to influence the extent and severity of the disease. In this study, the single nucleotide polymorphisms (SNPs) of tumor necrosis factor alpha (TNF-α) −308G/A (rs1800629) and interleukin-10 (IL-10) −1087G/A (rs1800896), −824C/T (rs1800871), and −597C/A (rs1800872) and the gamma interferon (IFN-γ) T/A SNP at position +874 (rs2430561) were typed in 80 Sicilian patients affected by MSF and in 288 control subjects matched for age, gender, and geographic origin. No significant differences in TNF-α −308G/A genotype frequencies were observed. The +874TT genotype, associated with an increased production of IFN-γ, was found to be significantly less frequent in MSF patients than in the control group (odds ratio [OR], 0.18; 95% confidence interval [95% CI], 0.06 to 0.51;Pcorrected for the number of genotypes [Pc], 0.0021). In addition, when evaluating the IFN-γ and IL-10 genotype interaction, a significant increase of +874AA/−597CA (OR, 5.31; 95% CI, 2.37 to 11.88;Pc, 0.0027) combined genotypes was observed. In conclusion, our data strongly suggest that finely genetically tuned cytokine production may play a crucial role in the regulation of the immune response against rickettsial infection, therefore influencing the disease outcomes, ranging from nonapparent or subclinical condition to overt or fatal disease.
Cell Division in
Escherichia coli
: Role of FtsL Domains in Septal Localization, Function, and Oligomerization
Authors: Ghigo Jean-Marc , Beckwith Jon
Published: April 2009

In
Escherichia coli
, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its
Haemophilus influenzae
homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.
Role of HrcA and CIRCE in the Heat Shock Regulatory Network of
Bradyrhizobium japonicum
Authors: Minder Alexander C. , Fischer Hans-Martin , Hennecke Hauke , Narberhaus Franz
Published: April 2009

A large number of bacteria regulate chaperone gene expression by the CIRCE-HrcA system in which a DNA element called CIRCE serves as binding site for the repressor protein HrcA under non-heat-shock conditions. We have cloned the two consecutive genes
hrcA
and
grpE
of
Bradyrhizobium japonicum
by using a complementation approach that screened for GrpE function. In vivo and in vitro transcript mapping demonstrated that both genes are transcribed separately from RpoH (ς
32
)-dependent promoters. To investigate the supposed negative regulatory function of HrcA, we compared the expression of putative target genes in the wild type with that in an
hrcA
mutant. Transcription of the CIRCE-associated chaperonin operons
groESL
4
and
groESL
5
, as well as the β-galactosidase activity derived from corresponding
groE-lacZ
fusions, was strongly elevated in the
hrcA
mutant even at physiological temperatures. Expression of other heat shock regulons (RpoH or ROSE dependent) was not affected. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein under nondenaturing conditions. Specific binding to the CIRCE element was obtained with a soluble fraction of HrcA in gel retardation experiments.
The Replicator of the Nopaline-Type Ti Plasmid pTiC58 Is a Member of the
repABC
Family and Is Influenced by the TraR-Dependent Quorum-Sensing Regulatory System
Authors: Li Pei-Li , Farrand Stephen K.
Published: April 2009

The replicator (
rep
) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the
trb
operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034–1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the
rep
locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the
traI-trb
operon, are closely related to the
repA
,
repB
, and
repC
genes of the octopine-type Ti plasmid pTiB6S3 as well as to other
repA
, -
B
, and -
C
genes from the Ri plasmid pRiA4b and three large plasmids from
Rhizobium
spp. The fourth ORF and the partial ORF are similar to
y4CG
and
y4CF
, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region between
traI
and
repA
contained two copies of the
tra
box which is the
cis
promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the
traI-trb
operon from the
tra
box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact
tra
box III. On the other hand, the region containing the two
tra
boxes was required for maximal expression of
repA
, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal
rep
plasmid increased five- to sevenfold in strains expressing
traR
but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.
Active Fe-Containing Superoxide Dismutase and Abundant
sodF
mRNA in
Nostoc commune
(Cyanobacteria) after Years of Desiccation
Authors: Shirkey Breanne , Kovarcik Don Paul , Wright Deborah J. , Wilmoth Gabriel , Prickett Todd F. , Helm Richard F. , Gregory Eugene M. , Potts Malcolm
Published: April 2009

Active Fe-superoxide dismutase (SodF) was the third most abundant soluble protein in cells of
Nostoc commune
CHEN/1986 after prolonged (13 years) storage in the desiccated state. Upon rehydration, Fe-containing superoxide disumutase (Fe-SOD) was released and the activity was distributed between rehydrating cells and the extracellular fluid. The 21-kDa Fe-SOD polypeptide was purified, the N terminus was sequenced, and the data were used to isolate
sodF
from the clonal isolate
N. commune
DRH1.
sodF
encodes an open reading frame of 200 codons and is expressed as a monocistronic transcript (of approximately 750 bases) from a region of the genome which includes genes involved in nucleic acid synthesis and repair, including dipyrimidine photolyase (
phr
) and cytidylate monophosphate kinase (
panC
).
sodF
mRNA was abundant and stable in cells after long-term desiccation. Upon rehydration of desiccated cells, there was a turnover of
sodF
mRNA within 15 min and then a rise in the mRNA pool to control levels (quantity of
sodF
mRNA in cells in late logarithmic phase of growth) over approximately 24 h. The extensive extracellular polysaccharide (glycan) of
N. commune
DRH1 generated superoxide radicals upon exposure to UV-A or -B irradiation, and these were scavenged by SOD. Despite demonstrated roles for the glycan in the desiccation tolerance of
N. commune
, it may in fact be a significant source of damaging free radicals in vivo. It is proposed that the high levels of SodF in
N. commune
, and release of the enzyme from dried cells upon rehydration, counter the effects of oxidative stress imposed by multiple cycles of desiccation and rehydration during UV-A or -B irradiation in situ.
Localized Reversible Frameshift Mutation in the
flhA
Gene Confers Phase Variability to Flagellin Gene Expression in
Campylobacter coli
Authors: Park Simon F. , Purdy Desmond , Leach Stephen
Published: April 2009

Phase variation of flagellin gene expression in
Campylobacter coli
UA585 was correlated with high-frequency, reversible insertion and deletion frameshift mutations in a short homopolymeric tract of thymine residues located in the N-terminal coding region of the
flhA
gene. Mutation-based phase variation in
flhA
may generate functional diversity in the host and environment.
Lesions in the
nuo
Operon, Encoding NADH Dehydrogenase Complex I, Prevent PurF-Independent Thiamine Synthesis and Reduce Flux through the Oxidative Pentose Phosphate Pathway in
Salmonella enterica
Serovar Typhimurium
Authors: Claas Kathy , Weber Shara , Downs Diana M.
Published: April 2009

In
Salmonella enterica
serovar Typhimurium, PurF-independent thiamine synthesis (or alternative pyrimidine biosynthesis) allows strains, under some growth conditions, to synthesize thiamine in the absence of the first step in the purine biosynthetic pathway. Mutations have been isolated in a number of loci that prevent this synthesis and thus result in an Apb
−
phenotype. Here we identify a new class of mutations that prevent PurF-independent thiamine synthesis and show that they are defective in the
nuo
genes, which encode the major, energy-generating NADH dehydrogenase of the cell. Data presented here indicated that a
nuo
mutant has reduced flux through the oxidative pentose phosphate pathway that may contribute to, but is not sufficient to cause, the observed thiamine requirement. We suggest that reduction of the oxidative pentose phosphate pathway capacity in a
nuo
mutant is an attempt to restore the ratio between reduced and oxidized pyridine nucleotide pools.
The
engL
Gene Cluster of
Clostridium cellulovorans
Contains a Gene for Cellulosomal ManA
Authors: Tamaru Yutaka , Doi Roy H.
Published: April 2009

A five-gene cluster around the gene in
Clostridium cellulovorans
that encodes endoglucanase EngL, which is involved in plant cell wall degradation, has been cloned and sequenced. As a result, a mannanase gene,
manA
, has been found downstream of
engL
. The
manA
gene consists of an open reading frame with 1,275 nucleotides encoding a protein with 425 amino acids and a molecular weight of 47,156. ManA has a signal peptide followed by a duplicated sequence (DS, or dockerin) at its N terminus and a catalytic domain which belongs to family 5 of the glycosyl hydrolases and shows high sequence similarity with fungal mannanases, such as
Agaricus bisporus
Cel4 (17.3% identity),
Aspergillus aculeatus
Man1 (23.7% identity), and
Trichoderma reesei
Man1 (22.7% identity). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequence analyses of the purified recombinant ManA (rManA) indicated that the N-terminal region of the rManA contained a DS and was truncated in
Escherichia coli
cells. Furthermore, Western blot analysis indicated that ManA is one of the cellulosomal subunits. ManA production is repressed by cellobiose.
O
-Acetylserine Sulfhydrylase from
Methanosarcina thermophila
Authors: Borup Birthe , Ferry James G.
Published: April 2009

Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the
Bacteria
and
Eucarya
domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the
Archaea
fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to either
O
-acetyl-
l
-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown
Methanosarcina thermophila
, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5′-phosphate and catalyzed the formation of
l
-cysteine and acetate from
O
-acetyl-
l
-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the
Eucarya
and
Bacteria
domains. The purified OASS had a specific activity of 129 μmol of cysteine/min/mg, with a

K
m

of 500 ± 80 μM for sulfide, and exhibited positive cooperativity and substrate inhibition with
O
-acetyl-
l
-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60°C, consistent with the optimum growth temperature for
M. thermophila
. The results of this study provide the first evidence for a sulfur-fixing enzyme in the
Archaea
domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the
Archaea
.
Listeria monocytogenes relAandhptMutants Are Impaired in Surface-Attached Growth and Virulence
Authors: Taylor Clare M. , Beresford Mark , Epton Harry A. S. , Sigee David C. , Shama Gilbert , Andrew Peter W. , Roberts Ian S.
Published: April 2009

We describe here the identification and characterization of twoListeria monocytogenes(Tn917-LTV3)relAandhpttransposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of therelAgene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in thehptmutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth ofL. monocytogenesin the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation ofL. monocytogenesfor biofilm growth and pathogenesis.
Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins
Authors: Nieto J. M. , Madrid C. , Miquelay E. , Parra J. L. , Rodríguez S. , Juárez A.
Published: April 2009

Escherichia coli
nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from
Yersinia enterocolitica
. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni
2+
-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of
Y. enterocolitica
. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in
Y. enterocolitica
, and found that it interacts with
Y. enterocolitica
H-NS. We also cloned and sequenced the
hns
gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from
E. coli
. We also found that the
hns
gene of
Y. enterocolitica
can complement an
hns
mutation of
E. coli
. Finally, we describe for the first time systematic characterization of missense mutant alleles of
hha
and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.
Novel Type of ADP-Forming Acetyl Coenzyme A Synthetase in Hyperthermophilic
Archaea
: Heterologous Expression and Characterization of Isoenzymes from the Sulfate Reducer
Archaeoglobus fulgidus
and the Methanogen
Methanococcus jannaschii
Authors: Musfeldt Meike , Schönheit Peter
Published: April 2009

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P
i
⇌ acetate + ATP + CoA) in
Archaea
and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile
Pyrococcus furiosus
, constitute 145-kDa heterotetramers (α
2
, β
2
). The coding genes for the α and β subunits are located at different sites in the
P. furiosus
chromosome. Based on significant sequence similarity of the
P. furiosus
genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon
Archaeoglobus fulgidus
and one ORF was identified in the hyperthermophilic methanogen
Methanococcus jannaschii
. The ORFs constitute fusions of the homologous
P. furiosus
genes encoding the α and β subunits. Two ORFs, AF1211 and AF1938, of
A. fulgidus
and ORF MJ0590 of
M. jannaschii
were cloned and functionally overexpressed in
Escherichia coli
. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the
Pyrococcus
ACD, the ACDs of
Archaeoglobus
and
Methanococcus
constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous
P. furiosus
α and β subunits in an αβ (AF1211 and MJ0590) or βα (AF1938) orientation. The data indicate that
A. fulgidus
and
M. jannaschii
contains a novel type of ADP-forming acetyl-CoA synthetase in
Archaea
, in which the subunit polypeptides and their coding genes are fused.
Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of
Desulfovibrio vulgaris
Hildenborough on Hydrogen and Lactate Metabolism
Authors: Pohorelic Brant K. J. , Voordouw Johanna K. , Lojou Elisabeth , Dolla Alain , Harder Jens , Voordouw Gerrit
Published: April 2009

The physiological properties of a
hyd
mutant of
Desulfovibrio vulgaris
Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of
D. vulgaris
Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The
hyd
mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H
2
as the sole electron donor, especially at a high sulfate concentration. Although the
hyd
mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H
2
was also present, growth in lactate- and sulfate-containing media lacking H
2
was less efficient. The
hyd
mutant produced, transiently, significant amounts of H
2
under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688–3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the
D. vulgaris
genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.
Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon
Aeropyrum pernix
: Interactions with the Two DNA Polymerases
Authors: Daimon Katsuya , Kawarabayasi Yutaka , Kikuchi Hisashi , Sako Yoshihiko , Ishino Yoshizumi
Published: April 2009

Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain
Eucarya
. We identified three genes encoding PCNA-like sequences in the genome of
Aeropyrum pernix
, a crenarchaeal archaeon. We cloned and expressed these genes in
Escherichia coli
and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in
A. pernix
to various extents, among which
A. pernix
PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the
A. pernix
cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in
A. pernix
cells under different conditions. In
Eucarya
, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.
Identification of the Periplasmic Cobalamin-Binding Protein BtuF ofEscherichia coli
Authors: Cadieux Nathalie , Bradbeer Clive , Reeger-Schneider Eva , Köster Wolfgang , Mohanty Arun K. , Wiener Michael C. , Kadner Robert J.
Published: April 2009

Cells ofEscherichia colitake up vitamin B12(cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to thebtuCEDoperon. The open reading frame termedyadTin theE. coligenomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake inSalmonella. TheE. coli yadTproduct, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His6tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation inbtuF, but not in the flanking genespfsandyadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of thebtuFmutant was much stronger than the slight impairment previously described forbtuC,btuD, orbtuFmutants. Hence, null mutations inbtuCandbtuDwere constructed and revealed that thebtuCmutant had a strong impairment similar to that of thebtuFmutant, whereas thebtuDdefect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.
Isolation and Characterization of BTF-37: Chromosomal DNA Captured from
Bacteroides fragilis
That Confers Self-Transferability and Expresses a Pilus-Like Structure in
Bacteroides
spp. and
Escherichia coli
Authors: Vedantam Gayatri , Hecht David W.
Published: April 2009

We report the isolation and preliminary characterization of BTF-37, a new 52-kb transfer factor isolated from
Bacteroides fragilis
clinical isolate LV23. BTF-37 was obtained by the capture of new DNA in the nonmobilizable
Bacteroides-Escherichia coli
shuttle vector pGAT400ΔBglII using a functional assay. BTF-37 is self-transferable within and from
Bacteroides
and also self-transfers in
E. coli
. Partial DNA sequencing, colony hybridization, and PCR revealed the presence of Tet element-specific sequences in BTF-37. In addition, Tn
5520
, a small mobilizable transposon that we described previously (G. Vedantam, T. J. Novicki, and D. W. Hecht, J. Bacteriol. 181:2564–2571, 1999), was also coisolated within BTF-37. Scanning and transmission electron microscopy of Tet element-containing
Bacteroides
spp. and BTF-37-harboring
Bacteroides
and
E. coli
strains revealed the presence of pilus-like cell surface structures. These structures were visualized in
Bacteroides
spp. only when BTF-37 and Tet element strains were induced with subinhibitory concentrations of tetracycline and resembled those encoded by
E. coli
broad-host-range plasmids. We conclude that we have captured a new, self-transferable transfer factor from
B. fragilis
LV23 and that this new factor encodes a tetracycline-inducible
Bacteroides
sp. conjugation apparatus.
Integration of Global Regulation of Two Aromatic-Responsive σ54-Dependent Systems: a Common Phenotype by Different Mechanisms
Authors: Sze Chun Chau , Bernardo Lisandro M. D. , Shingler Victoria
Published: April 2009

Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related σ54-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding σ54RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of σ54-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the nativePseudomonas putidaand a heterologousEscherichia colihost. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.