bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of New Hydrogen-Recycling
Rhizobiaceae
Strains by Introduction of a Novel
hup
Minitransposon
Authors: Báscones Elena , Imperial Juan , Ruiz-Argüeso Tomás , Palacios Jose Manuel
Published: July 2002

Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the
Rhizobium
-legume symbiosis. To develop a strategy to generate rhizobial strains with H
2
-recycling ability, we have constructed a Tn
5
derivative minitransposon (TnHB100) that contains the ca. 18-kb H
2
uptake (
hup
) gene cluster from
Rhizobium leguminosarum
bv. viciae UPM791. Bacteroids from TnHB100-containing strains of
R. leguminosarum
bv. viciae PRE,
Bradyrhizobium japonicum
,
R. etli
, and
Mesorhizobium loti
expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules. Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast,
Sinorhizobium meliloti
,
M. ciceri
, and
R. leguminosarum
bv. viciae UML2 strains showed poor expression of the
hup
system that resulted in H
2
-evolving nodules. For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of
hup
genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release.
A Food-Grade Cloning System for Industrial Strains of
Lactococcus lactis
Authors: Sørensen Kim I. , Larsen Rasmus , Kibenich Annette , Junge Mette P. , Johansen Eric
Published: July 2002

We have previously reported the construction of a food-grade cloning vector for
Lactococcus
using the ochre suppressor,
supB
, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As
supB
suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of
Lactococcus lactis
. The vector uses an amber suppressor,
supD
, as selectable marker and consists entirely of
Lactococcus
DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant
L. lactis
strains. The usefulness of this system is demonstrated by overexpressing the
pepN
gene in a number of industrial backgrounds.
Magnetic Bead Capture Eliminates PCR Inhibitors in Samples Collected from the Airborne Environment, Permitting Detection of
Pneumocystis carinii
DNA
Authors: Maher Nancy , Dillon H. Kenneth , Vermund Sten H. , Unnasch Thomas R.
Published: July 2002

PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S
Pneumocystis carinii
mitochondrial rRNA gene-based PCR.
Survival Response and Rearrangement of Plasmid DNA of
Lactococcus lactis
during Long-Term Starvation
Authors: Kim Woojin S. , Park Ji Hyeon , Ren Jun , Su Ping , Dunn Noel W.
Published: July 2002

The survival response of
Lactococcus lactis
during long-term starvation was investigated. The cells were cultured with different levels of glucose (the sole energy source) and either were kept in the resultant spent medium or transferred to fresh medium (without glucose) for up to 2 years. The survival of the cells during starvation was not dependent on the nature of transition phase, as expected, but on the nature of medium in which the cells were kept. The proliferation of cells, despite the apparent lack of glucose, could have been due to some cells being able to utilize the small amounts of peptides still present in the spent medium or to use energy sources provided by the breakup of dead cells. The 1- and 2-year-old cultures contained cells with vastly changed morphotypes. When these isolates were examined, it was revealed that the original plasmids present in the parent were rearranged in a certain way, and an entirely new plasmid was generated. Changes were also evident in the chromosomal DNA and in gene expression. Furthermore, all of the isolates exhibited a growth advantage relative to the parent cells when grown in energy-limiting media. When they were tested against different types of stresses, they exhibited a higher resistance against the bile salt and hydrogen peroxide stresses compared to the parent. Because of the similar changes observed in the 2-year-old isolates, a similar survival strategy may be operational in those cells that survive for that length of time.
Novel
psbA1
Gene from a Naturally Occurring Atrazine-Resistant Cyanobacterial Isolate
Authors: Sajjaphan Kannika , Shapir Nir , Judd Adam K. , Wackett Lawrence P. , Sadowsky Michael J.
Published: July 2002

A naturally occurring atrazine-resistant cyanobacterial isolate, strain SG2, was isolated from an atrazine-containing wastewater treatment system at the Syngenta atrazine production facility in St. Gabriel, La. Strain SG2 was resistant to 1,000 μg of atrazine per ml but showed relatively low resistance to diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl urea]. Analyses of 16S ribosomal DNA indicated that strain SG2 falls into the
Synechocystis
/
Pleurocapsa
/
Microcystis
group. Photosynthetically driven oxygen evolution in strain SG2 was only slightly inhibited (about 10%) by 2,000 μg of atrazine per ml, whereas in the control strain
Synechocystis
6803, oxygen evolution was inhibited 90% by 1,000 μg of atrazine per ml. No atrazine accretion, mineralization, or metabolites were detected when strain SG2 was grown with [
14
C]atrazine. Strain SG2 contained three copies of the
psbA
gene, which encodes the D
1
protein of the photosystem II reaction center. Nucleotide sequence analyses indicated that the
psbA2
and
psbA3
genes encoded predicted proteins with the same amino acid sequence. However, the
psbA1
gene product contained five extra amino acids, which were not found in PsbA proteins from five other cyanobacteria. Moreover, the PsbA1 protein from strain SG2 had an additional 13 amino acid changes compared to the PsbA2/PsbA3 proteins and contained 10 amino acid alterations compared to conserved residues found in other cyanobacteria. Reverse transcriptase PCR analysis indicated that the
psbA1
gene and the
psbA2
/
psbA3
gene(s) were expressed in photosynthetically grown cells in the presence of atrazine. These results suggest that strong selection pressure conferred by the continual input of atrazine has contributed to the evolution of a herbicide-resistant, yet photosynthetically efficient,
psbA
gene in a cyanobacterium.
Spatial Distribution of Total, Ammonia-Oxidizing, and Denitrifying Bacteria in Biological Wastewater Treatment Reactors for Bioregenerative Life Support
Authors: Sakano Yuko , Pickering Karen D. , Strom Peter F. , Kerkhof Lee J.
Published: July 2002

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project—Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (
amoA
) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (
nosZ
) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of
Proteobacteria
, low-G+C gram-positive bacteria, and a
Cytophaga-Flexibacter-Bacteroides
group. Fifty-seven novel 16S rRNA genes, 8 novel
amoA
genes, and 12 new
nosZ
genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.
Anti-Idiotypic Antibodies in Patients with Different Clinical Forms of Paracoccidioidomycosis
Authors: Souza A. R. , Gesztesi J.-L. , del Negro G. M. B. , Benard G. , Sato J. , Santos M. V. B. , Abrahão T. B. , Lopes J. D.
Published: July 2002

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors. Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form. The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43). All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43. Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system. The possible correlation of Ab2 titers with different clinical forms of disease was also verified. Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a). Spontaneous anti-Id antibodies were found in all clinical forms of disease. The majority of patients (88%,n= 8) with the acute form of PCM had high titers of Ab2. However, among patients with the multifocal chronic form of the disease, only 29% (n= 14) had high titers of Ab2; 70% (n= 10) of patients with the unifocal chronic form had low titers of Ab2. A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment. Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.
Serodiagnostic Potential of Culture Filtrate Antigens of
Mycobacterium tuberculosis
Authors: Samanich K. M. , Keen M. A. , Vissa V. D. , Harder J. D. , Spencer J. S. , Belisle J. T. , Zolla-Pazner S. , Laal S.
Published: July 2002

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of
Mycobacterium tuberculosis
that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534–1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49–56, 1997), in which all sera are preadsorbed against
Escherichia coli
lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in
E. coli
, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.
Antibody Responses to
Campylobacter
Infections Determined by an Enzyme-Linked Immunosorbent Assay: 2-Year Follow-Up Study of 210 Patients
Authors: Strid Mette Aagaard , Engberg Jørgen , Larsen Lena Brandt , Begtrup Kamilla , Mølbak Kåre , Krogfelt Karen Angeliki
Published: July 2002

An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to
Campylobacter jejuni
and
Campylobacter coli
. Heat-stable antigen, a combination of
C. jejuni
serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified
Campylobacter
enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on
Campylobacter
species or
C. jejuni
serotype, with the important exception of response to
C. jejuni
O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (
P
= 0.006) and IgA (
P
= 0.06) compared with other
C. jejuni
and
C. coli
serotypes.
Self Antigen Prognostic for Human Immunodeficiency Virus Disease Progression
Authors: Bristow Cynthia L. , Patel Hirenkumar , Arnold Roland R.
Published: July 2002

We have recently found that an extracellular protein, α1proteinase inhibitor (α1PI; α1antitrypsin), is required for in vitro human immunodeficiency virus (HIV) infectivity outcome. We show here in a study of HIV-seropositive patients that decreased viral load is significantly correlated with decreased circulating α1PI. In the asymptomatic category of HIV disease, 100% of patients manifest deficient levels of active α1PI, a condition known to lead to degenerative lung diseases and a dramatically reduced life span. Further, HIV-associated α1PI deficiency is correlated with circulating anti-α1PI immunoglobulin G. These results suggest that preventing HIV-associated α1PI deficiency may provide a strategic target for preventing HIV-associated pathophysiology.
Identification and Characterization of Murine Cytotoxic T Cells That KillMycobacterium tuberculosis
Authors: Silva Celio L. , Lowrie Douglas B.
Published: July 2002

As we seek to develop and evaluate new vaccines against tuberculosis, it is desirable that we understand the mechanisms of protective immunity in our models. Adoptive transfer of protection with hsp65-specific T-cell clones from infected or vaccinated mice into naı̈ve mice had indicated that cytotoxic T cells can make a major contribution to protection. We characterized 28 CD4+CD8−and 28 CD4−CD8+hsp65-specific T-cell clones derived from infected or vaccinated mice. Half of the CD4+CD8−and 64% of the CD4−CD8+clones were cytotoxic. Cytotoxicity was associated with high expression of CD44 and gamma interferon production. Most (86%) of the cytotoxic CD4+CD8−clones lysed target cells via the Fas-FasL pathway, and most (83%) of the cytotoxic CD4−CD8+clones lysed target cells via cytotoxic granules. Only the clones using the granule-mediated pathway caused substantial loss of viability of virulentMycobacterium tuberculosisduring lysis of infected macrophages, and the degree of killing closely correlated with the availability of granule marker enzyme activity. Granule-mediated cytotoxicity thus may have a key role in protection against tuberculosis by delivering mycobactericidal granule contents.
Guillain-Barré Syndrome- and Miller Fisher Syndrome-Associated
Campylobacter jejuni
Lipopolysaccharides Induce Anti-GM
1
and Anti-GQ
1b
Antibodies in Rabbits
Authors: Ang C. W. , De Klerk M. A. , Endtz H. P. , Jacobs B. C. , Laman J. D. , van der Meché F. G. A. , van Doorn P. A.
Published: July 2002

Campylobacter jejuni
infections are thought to induce antiganglioside antibodies in patients with Guillain-Barrésyndrome (GBS) and Miller Fisher syndrome (MFS) by molecular mimicry between
C. jejuni
lipopolysaccharides (LPS) and gangliosides. We used purified LPS fractions from five
Campylobacter
strains to induce antiganglioside responses in rabbits. The animals that received injections with LPS from GBS-associated strains developed anti-GM
1
and anti-GA
1
antibodies. Animals injected with LPS from one MFS-related
C. jejuni
strain produced anti-GQ
1b
antibodies. Rabbits that were injected with Penner O:3 LPS had a strong anti-LPS response, but no antiganglioside reactivity was observed. The antiganglioside specificity in the rabbits reflected the specificity in the patients from whom the strains were isolated. In conclusion, our results indicate that an immune response against GBS- and MFS-associated
C. jejuni
LPS results in antiganglioside antibodies. These results provide strong support for molecular mimicry as a mechanism in the induction of antiganglioside antibodies following infections.
Assessing Vascular Permeability during Experimental Cerebral Malaria by a Radiolabeled Monoclonal Antibody Technique
Authors: van der Heyde Henri C. , Bauer Philippe , Sun Guang , Chang Wun-Ling , Yin Lijia , Fuseler John , Granger D. Neil
Published: July 2002

Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly compromised in the brain, lung, kidney, and heart duringPlasmodium bergheiinfection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were significantly (P< 0.001) related.
Polymorphisms in Pilin Glycosylation Locus of
Neisseria meningitidis
Expressing Class II Pili
Authors: Kahler Charlene M. , Martin Larry E. , Tzeng Yih-Ling , Miller Yoon K. , Sharkey Kerith , Stephens David S. , Davies John K.
Published: July 2002

We have located a locus,
pgl
, in
Neisseria meningitidis
strain NMB required for the glycosylation of class II pili. Between five and eight open reading frames (ORFs) (
pglF, pglB, pglC, pglB2, orf2, orf3, orf8
, and
avtA
) were present in the
pgl
clusters of different meningococcal isolates. The Class I pilus-expressing strains
Neisseria gonorrhoeae
MS11 and
N. meningitidis
MC58 each contain a
pgl
cluster in which
orf2
and
orf3
have been deleted. Strain NMB and other meningococcal isolates which express class II type IV pili contained
pgl
clusters in which
pglB
had been replaced by
pglB2
and an additional novel ORF,
orf8
, had been inserted between
pglB2
and
pglC
. Insertional inactivation of the eight ORFs of the
pgl
cluster of strain NMB showed that
pglF, pglB2, pglC
, and
pglD,
but not
orf2, orf3, orf8
, and
avtA
, were necessary for pilin glycosylation. Pilin glycosylation was not essential for resistance to normal human serum, as
pglF
and
pglD
mutants retained wild-type levels of serum resistance. Although
pglB2
and
pglC
mutants were significantly sensitive to normal human serum under the experimental conditions used, subsequent examination of the encapsulation phenotypes revealed that
pglB2
and
pglC
mutants expressed almost 50% less capsule than wild-type NMB. A mutation in
orf3
, which did not affect pilin glycosylation, also resulted in a 10% reduction in capsule expression and a moderately serum sensitive phenotype. On the basis of these results we suggest that pilin glycosylation may proceed via a lipid-linked oligosaccharide intermediate and that blockages in this pathway may interfere with capsular transport or assembly.
Variance in Fibronectin Binding and
fnb
Locus Polymorphisms in
Staphylococcus aureus
: Identification of Antigenic Variation in a Fibronectin Binding Protein Adhesin of the Epidemic CMRSA-1 Strain of Methicillin-Resistant
S. aureus
Authors: Rice Kelly , Huesca Mario , Vaz Dareyl , McGavin Martin J.
Published: July 2002

The
fnbA
and
fnbB
genes of
Staphylococcus aureus
8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of
fnb
genes or the number of D motifs, we identified five polymorphism groups.
S. aureus
8325-4 and 91% of methicillin-resistant
S. aureus
(MRSA) isolates belong to polymorphism group I, with two
fnb
genes and three copies of the D motif. Polymorphism group II contained one
fnb
gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number of
fnb
genes or the number of D motifs. Therefore, the
fnb
locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.
Effect of Purified Lipopolysaccharides from Strains of
Helicobacter pylori
and
Helicobacter felis
on Acid Secretion in Mouse Gastric Glands In Vitro
Authors: Padol Ireneusz T. , Moran Anthony P. , Hunt Richard H.
Published: July 2002

As a bacterial product,
Helicobacter pylori
lipopolysaccharide (LPS) can originate in close proximity to parietal cells, but the role of this uniquely structured endotoxin on acid secretion has not been fully investigated and remains unclear. The purpose of this study was to test the direct effect of purified LPS (tested range, 0.1 to 100 μg/ml) from various strains of
H. pylori
and from one
Helicobacter felis
strain on histamine- and carbachol-stimulated acid secretion in vitro using mouse gastric glands and the accumulation of [
14
C]aminopyrine. In addition, we investigated whether
H. pylori
LPS can interfere with two native antisecretory substances, prostaglandin E
2
(PGE
2
) and somatostatin, which may contribute to bacterial pathogenicity. Except for the LPS from
H. pylori
SS1 (Sydney strain), which gave a statistically significant increase in both histamine- and carbachol-stimulated acid output (38 and 24%, respectively;
P
< 0.05), no effect of the tested LPS was observed on acid secretion.
H. pylori
LPS purified from a patient isolate did not affect the potency or the efficacy of the inhibitory dose response curve to PGE
2
or somatostatin. Bacterial interstrain variation in the direct stimulatory effect of
Helicobacter
-derived LPS on acid secretion was observed, which probably reflects the molecular structure of LPS and the potential to contribute to virulence. Importantly, the data showed that
H. pylori
LPS did not have any direct antisecretory properties. It can be speculated that the acid stimulatory properties of LPS from
H. pylori
SS1 may contribute to the gastric damage observed in the mouse model of
H. pylori
infection.
γ-Glutamyltransferase Is a
Helicobacter pylori
Virulence Factor but Is Not Essential for Colonization
Authors: McGovern K. J. , Blanchard T. G. , Gutierrez J. A. , Czinn S. J. , Krakowka S. , Youngman P.
Published: July 2002

The contribution of glutamyl transpeptidase (GGT) (γ-glutamyltransferase [EC 2. 3. 2. 2]) to
Helicobacter pylori
virulence was investigated in piglets and mice using GGT-deficient isogenic strains. All animals became colonized. However, the bacterial load was significantly lower for mutant bacteria than for parent strains. These results suggest that GGT activity provides an advantage to
H. pylori
in colonization.
Virulence Gene Identification by Differential Fluorescence Induction Analysis of
Staphylococcus aureus
Gene Expression during Infection-Simulating Culture
Authors: Schneider William P. , Ho Sun K. , Christine Jillian , Yao Monique , Marra Andrea , Hromockyj Alexander E.
Published: July 2002

We have employed a strategy utilizing differential fluorescence induction (DFI) in an effort to identify
Staphylococcus aureus
genes whose products can be targeted for antimicrobial drug development. DFI allows identification of promoters preferentially active under given growth conditions on the basis of their ability to drive expression of a promoterless green fluorescent protein gene (
gfp
). A plasmid-based promoter trap library was constructed of 200- to 1,000-bp fragments of
S. aureus
genomic DNA fused to
gfp
, and clones with active promoters were isolated under seven different in vitro growth conditions simulating infection. Six thousand two hundred sixty-seven clones with active promoters were screened to identify those that exhibited differential promoter activity. Bioinformatic analysis allowed the identification of 42 unique operons, containing a total of 61 genes, immediately downstream of the differentially active putative promoters. Replacement mutations were generated for most of these operons, and the abilities of the resulting mutants to cause infection were assessed in two different murine infection models. Approximately 40% of the mutants were attenuated in at least one infection model.
Complementation of a
Treponema denticola flgE
Mutant with a Novel Coumermycin A1-Resistant
T. denticola
Shuttle Vector System
Authors: Chi Bo , Limberger Ronald J. , Kuramitsu Howard K.
Published: July 2002

By using the mutated
gyrB
gene from a spontaneous coumermycin A1-resistant
Treponema denticola
, an
Escherichia coli-T. denticola
shuttle vector that renders
T. denticola
resistant to coumermycin was constructed. The complete
T. denticola flgE
gene was cloned into the shuttle vector pKMCou, and the vector was transformed into the
T. denticola
ATCC 33520
flgE
erythromycin-resistant knockout mutant HL210. The coumermycin-resistant transformants were motile and restored FlgE activity. This complementation system should prove useful in studying the virulence factors of
T. denticola
and uncultivable pathogenic spirochetes.
Genome Sequence Survey Identifies Unique Sequences and Key Virulence Genes with Unusual Rates of Amino Acid Substitution in Bovine
Staphylococcus aureus
Authors: Herron Lisa L. , Chakravarty Rajit , Dwan Christopher , Fitzgerald J. Ross , Musser James M. , Retzel Ernest , Kapur Vivek
Published: July 2002

Staphylococcus aureus
is a major cause of mastitis in bovine and other ruminant species. We here present the results of a comparative genomic analysis between a bovine mastitis-associated clone, RF122, and the recently sequenced human-associated clones, Mu50 and N315, of
Staphylococcus aureus
. A shotgun sequence survey of ∼10% of the RF122 genome identified numerous unique sequences and those with elevated rates of nonsynonymous substitution. Taken together, these analyses show that there are notable differences in the genomes of bovine mastitis-associated and human clones of
S. aureus
and provide a framework for the identification of specific factors associated with host specificity in this major human and animal pathogen.
Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding
Authors: Cho Eun Hee , Alcaraz Renato , Gumport Richard I. , Gardner Jeffrey F.
Published: July 2002

The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.
Increased Expression of
Escherichia coli
Polynucleotide Phosphorylase at Low Temperatures Is Linked to a Decrease in the Efficiency of Autocontrol
Authors: Mathy N. , Jarrige A.-C. , Robert-Le Meur M. , Portier C.
Published: July 2002

Polynucleotide phosphorylase (PNPase) synthesis is translationally autocontrolled via an RNase III-dependent mechanism, which results in a tight correlation between protein level and messenger stability. In cells grown at 18°C, the amount of PNPase is twice that found in cells grown at 30°C. To investigate whether this effect was transcriptional or posttranscriptional, the expression of a set of
pnp-lacZ
transcriptional and translational fusions was analyzed in cells grown at different temperatures. In the absence of PNPase, there was no increase in
pnp-lacZ
expression, indicating that the increase in
pnp
expression occurs at a posttranscriptional level. Other experiments clearly show that increased
pnp
expression at low temperature is only observed under conditions in which the autocontrol mechanism of PNPase is functional. At low temperature, the destabilizing effect of PNPase on its own mRNA is less efficient, leading to a decrease in repression and an increase in the expression level.
Unusual Regulatory Elements for Iron Deficiency Induction of the
idiA
Gene of
Synechococcus elongatus
PCC 7942
Authors: Michel Klaus-Peter , Pistorius Elfriede K. , Golden Susan S.
Published: July 2002

Expression of a thylakoid membrane-associated protein called IdiA (
i
ron-
d
eficiency-
i
nduced protein
A
) is highly elevated and tightly regulated by iron limitation in
Synechococcus elongatus
PCC 6301 and PCC 7942. Although this protein is not essential for photosystem II (PSII) activity, it plays an important role in protecting the acceptor side of PSII against oxidative damage, especially under iron-limiting growth conditions, by an unknown mechanism. We defined the iron-responsive
idiA
promoter by using insertional inactivation mutagenesis and reporter gene assays. A 67-bp DNA region was sufficient for full iron deficiency-inducible
idiA
promoter activity. Within this fragment is a palindromic sequence 4 bp upstream of a putative −35 promoter element, which resembles the binding site of FNR/CAP-type helix-turn-helix transcription factors. The absence of this palindromic sequence or a 3-bp mutation in a putative −10 region eliminated promoter activity completely. A previously identified candidate for a positively acting transcription factor is the IdiB protein, whose gene lies immediately downstream of
idiA
. IdiB shows strong similarity to helix-turn-helix transcription factors of the FNR/CAP family. A His
6x
-tagged IdiB that was overexpressed in
Escherichia coli
bound to a 59-bp fragment of the
idiA
regulatory region that included the palindrome. Although the
idiA
promoter lacks a consensus binding site for the iron-sensing regulator Fur, we attempted to inactivate
fur
in order to investigate the potential role of this factor. The resulting merodiploid mutants showed constitutive partial derepression of IdiA expression under iron-sufficient growth conditions. We concluded that IdiB is a specific iron-responsive regulator of
idiA
and that Fur has an indirect role in influencing
idiA
expression.
Thermotoga maritima
Phosphofructokinases: Expression and Characterization of Two Unique Enzymes
Authors: Ding Yan-Huai R. , Ronimus Ron S. , Morgan Hugh W.
Published: July 2002

A pyrophosphate-dependent phosphofructokinase (PP
i
-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from
Thermotoga maritima
have been cloned and characterized. The PP
i
-PFK is unique in that the
K

m

and
V
max
values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP
i
and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.
Control of Nitrogenase Reactivation by the GlnZ Protein in
Azospirillum brasilense
Authors: Klassen Giseli , de Souza Emanuel M. , Yates M. Geoffrey , Rigo Liu Un , Inaba Juliana , de Oliveira Pedrosa Fábio
Published: July 2002

The
glnZ
mutant of
Azospirillum brasilense
(strain 7611) showed only partial recovery (20 to 40%) after 80 min of ammonia-induced nitrogenase switch-off, whereas the wild type recovered totally within 10 min. In contrast, the two strains showed identical anoxic-induced switch-on/switch-off, indicating no cross talk between the two reactivation mechanisms.
Genetic Screen Yields Mutations in Genes Encoding All Known Components of the
Escherichia coli
Signal Recognition Particle Pathway
Authors: Tian Hongping , Beckwith Jon
Published: July 2002

We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the
Escherichia coli
cytoplasmic membrane. The screen yielded mutations in each of the known genes encoding components of the
E. coli
signal recognition particle pathway:
ffh
,
ffs
, and
ftsY
, which encode Ffh, 4.5S RNA, and FtsY, respectively. In addition, the screen yielded mutations in
secM
, which is involved in regulating levels of the SecA component of the bacterium’s protein export pathway. We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ. Among the mutations that resulted in membrane protein insertion defects, only the
secM
mutations also showed defects in the translocation of proteins into the
E. coli
periplasm. Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.
In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host
Authors: Rose R. Wesley , Pohlschröder Mechthild
Published: July 2002

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (
hv54h
,
hv19h
, and
hv7Sh
, respectively) of the genetically and biochemically tractable archaeon
Haloferax volcanii
were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an
hv54h
knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in
H. volcanii
, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.
Nitric Oxide-Induced Homologous Recombination in
Escherichia coli
Is Promoted by DNA Glycosylases
Authors: Spek Erik J. , Vuong Laurel N. , Matsuguchi Tetsuya , Marinus Martin G. , Engelward Bevin P.
Published: July 2002

Nitric oxide (NO
.
) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO
.
leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO
.
toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO
.
toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO
.
-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in
recBCD
cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting
ung
and
fpg
suppressed NO
.
-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO
.
-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO
.
-induced homologous recombination.
Identification of Isolates ofStreptococcus canisInfecting Humans
Authors: Whatmore Adrian M. , Engler Kathryn H. , Gudmundsdottir Gudny , Efstratiou Androulla
Published: July 2002

During a survey of Group G and C streptococcal infections of humans two epidemiologically unrelated Group G streptococcal isolates were identified, one from a case of bacteremia and one from a wound infection. These isolates were atypical among this sample in that theemmgene could not be amplified from them by PCR. Biochemical characterization identified the isolates asStreptococcus canis, an organism normally associated with animal hosts. The biochemical identification was confirmed by sequencing of the 16S rRNA gene from both isolates and comparison with sequences of theS. canistype strain and other related streptococci of animals and humans. Comparative sequencing of fragments of two other housekeeping genes,sodAandmutS, confirmed that the isolates are most closely related toS. canis. The identification of two isolates ofS. canisfrom a relatively small sample set suggests that the practice of identifying streptococci only by the Lancefield serological group may result in underestimation of the presence ofS. canisin the human population.
Phospholipase Region of
Mycobacterium tuberculosis
Is a Preferential Locus for IS
6110
Transposition
Authors: Vera-Cabrera Lucio , Hernández-Vera Marco A. , Welsh Oliverio , Johnson Wendy M. , Castro-Garza Jorge
Published: July 2002

Enzymes with phospholipase C activity in
Mycobacterium tuberculosis
have been recently described. The three genes encoding these proteins,
plcA
,
plcB
, and
plcC
, are located at position 2351 of the genomic map of
M. tuberculosis
H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the
plcA
and
plcB
genes in
M. tuberculosis
clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic
M. tuberculosis
clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the
plcA
and
plcB
genes and part of the
plcC
gene. In the
M. tuberculosis
strains studied the production of an amplicon ∼1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS
6110
element. We observed insertion elements in the
plcA
,
plcB
, and
plcC
genes. One site in
plcB
had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in
plcA
in the same nucleotide position. In all the cases, IS
6110
was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS
6110
elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included
plcA
and most of
plcB
. This can explain the negative results obtained by some authors when detecting the
mtp40
sequence (
plcA
) by PCR. Given the high polymorphism in this region, the use of the
mtp40
sequence as a genetic marker for
M. tuberculosis
sensu stricto is very restricted.
Identification of Clinical Isolates of
Actinomyces
Species by Amplified 16S Ribosomal DNA Restriction Analysis
Authors: Hall Val , Talbot P. R. , Stubbs S. L. , Duerden B. I.
Published: July 2002

Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes
Hae
III and
Hpa
II, was applied to 176 fresh and 299 stored clinical isolates of putative
Actinomyces
spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized
Actinomyces
species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than
Actinomyces
. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of
Actinomyces
. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as
A. meyeri
or resembling
A. meyeri
by phenotypic tests, only 21 were confirmed as
A. meyeri
by ARDRA; 63 were reassigned as
A. turicensis
, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of
Actinomyces
spp. add to the currently sparse knowledge of some recently described species.
Differentiation of Resistance Phenotypes among Erythromycin-Resistant Pneumococci
Authors: Montanari Maria Pia , Mingoia Marina , Giovanetti Eleonora , Varaldo Pietro Emanuele
Published: July 2002

Laboratory differentiation of erythromycin resistance phenotypes is poorly standardized for pneumococci. In this study, 85 clinical isolates of erythromycin-resistant (MIC ≥ 1 μg/ml)
Streptococcus pneumoniae
were tested for the resistance phenotype by the erythromycin-clindamycin double-disk test (previously used to determine the macrolide resistance phenotype in
Streptococcus pyogenes
strains) and by MIC induction tests, i.e., by determining the MICs of macrolide antibiotics without and with pre-exposure to 0.05 μg of erythromycin per ml. By the double-disk test, 65 strains, all carrying the
erm
(AM) determinant, were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and the remaining 20, all carrying the
mef
(E) gene, were assigned to the recently described M phenotype; an inducible MLS resistance (iMLS) phenotype was not found. The lack of inducible resistance to clindamycin was confirmed by determining clindamycin MICs without and with pre-exposure to subinhibitory concentrations of erythromycin. In macrolide MIC and MIC-induction tests, whereas homogeneous susceptibility patterns were observed among the 20 strains assigned to the M phenotype by the double-disk test, two distinct patterns were recognized among the 65 strains assigned to the cMLS phenotype by the same test; one pattern (
n
= 10; probably that of the true cMLS isolates) was characterized by resistance to rokitamycin also without induction, and the other pattern (
n
= 55; designated the iMcLS phenotype) was characterized by full or intermediate susceptibility to rokitamycin without induction turning to resistance after induction, with an MIC increase by more than three dilutions. A triple-disk test, set up by adding a rokitamycin disk to the erythromycin and clindamycin disks of the double-disk test, allowed the easy differentiation not only of pneumococci with the M phenotype from those with MLS resistance but also, among the latter, of those of the true cMLS phenotype from those of the iMcLS phenotype. While distinguishing MLS from M resistance in pneumococci is easily and reliably achieved, the differentiation of constitutive from inducible MLS resistance is far more uncertain and is strongly affected by the antibiotic used to test inducibility.
Increasing Multidrug Resistance in
Helicobacter pylori
Strains Isolated from Children and Adults in Mexico
Authors: Torres Javier , Camorlinga-Ponce Margarita , Pérez-Pérez Guillermo , Madrazo-De la Garza Armando , Dehesa Margarita , González-Valencia Gerardo , Muñoz Onofre
Published: July 2002

The susceptibilities to three antimicrobials of 195
Helicobacter pylori
strains isolated from Mexican patients is reported; 80% of the strains were resistant to metronidazole, 24% were resistant to clarithromycin, and 18% presented a transient resistance to amoxicillin. Resistance to two or more antimicrobials increased significantly from 1995 to 1997.