bluuebunny/crossref_metadata_2025 huggingface dataset:
High Extracellular Levels of
Mycobacterium tuberculosis
Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism
Authors: Tullius Michael V. , Harth Günter , Horwitz Marcus A.
Published: July 2002

Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of
Mycobacterium tuberculosis
, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (
glnA1
and
sodA
) from the rapid-growing, nonpathogenic
Mycobacterium smegmatis
, generated
glnA1
and
sodA
mutants of
M. smegmatis
by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from
M. tuberculosis
. When each of the genes was expressed from a multicopy plasmid,
M. smegmatis
exported comparable proportions of both the
M. tuberculosis
and
M. smegmatis
GSs (in the
glnA1
strain) or SODs (in the
sodA
strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant
M. smegmatis
and
M. tuberculosis
strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant
M. tuberculosis
strain expressing green fluorescent protein (GFP) at high levels and a recombinant
M. smegmatis
strain coexpressing the
M. smegmatis
GS,
M. smegmatis
SOD, and
M. tuberculosis
BfrB (bacterioferritin) at high levels. The recombinant
M. tuberculosis
strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous
M. tuberculosis
GS and SOD. Similarly, the recombinant
M. smegmatis
strain exported bacterioferritin, a large (∼500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of
M. tuberculosis
GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing
M. tuberculosis
cultures.
Epitope Mapping of Neutralizing Botulinum Neurotoxin A Antibodies by Phage Display
Authors: Mullaney B. P. , Pallavicini M. G. , Marks J. D.
Published: July 2002

Single-chain antibodies neutralize activity and bind nonoverlapping epitopes of botulinum A neurotoxin. Two phage display epitope libraries were constructed from the 1.3 kb of binding domain cDNA. The minimal epitopes selected against the single-chain Fv-Fc antibodies correspond to conformational epitopes with amino acid residues 1115 to 1223 (S25), 1131 to 1264 (3D12), and 889 to 1294 (C25).
Salmonella enterica
Serovar Typhi Possesses a Unique Repertoire of Fimbrial Gene Sequences
Authors: Townsend Stacy M. , Kramer Naomi E. , Edwards Robert , Baker Stephen , Hamlin Nancy , Simmonds Mark , Stevens Kim , Maloy Stanley , Parkhill Julian , Dougan Gordon , Bäumler Andreas J.
Published: July 2002

Salmonella enterica
serotype Typhi differs from nontyphoidal
Salmonella
serotypes by its strict host adaptation to humans and higher primates. Since fimbriae have been implicated in host adaptation, we investigated whether the serotype Typhi genome contains fimbrial operons which are unique to this pathogen or restricted to typhoidal
Salmonella
serotypes. This study established for the first time the total number of fimbrial operons present in an individual
Salmonella
serotype. The serotype Typhi CT18 genome, which has been sequenced by the Typhi Sequencing Group at the Sanger Centre, contained a type IV fimbrial operon, an orthologue of the
agf
operon, and 12 putative fimbrial operons of the chaperone-usher assembly class. In addition to
sef, fim, saf
, and
tcf
, which had been described previously in serotype Typhi, we identified eight new putative chaperone-usher-dependent fimbrial operons, which were termed
bcf, sta, stb, ste, std, stc, stg
, and
sth
. Hybridization analysis performed with 16 strains of
Salmonella
reference collection C and 22 strains of
Salmonella
reference collection B showed that all eight putative fimbrial operons of serotype Typhi were also present in a number of nontyphoidal
Salmonella
serotypes. Thus, a simple correlation between host range and the presence of a single fimbrial operon seems at present unlikely. However, the serotype Typhi genome differed from that of all other
Salmonella
serotypes investigated in that it contained a unique combination of putative fimbrial operons.
Contribution of CD8+T Cells to Gamma Interferon Production in Human Tuberculosis
Authors: Shams Homayoun , Wizel Benjamin , Weis Stephen E. , Samten Buka , Barnes Peter F.
Published: July 2002

The proportions of peripheral blood mononuclear cells (PBMC), CD4+T cells, and CD8+T cells that produce gamma interferon (IFN-γ) in response toMycobacterium tuberculosiswere markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4+but not CD8+cells prior to stimulation of PBMC withM. tuberculosisabolished IFN-γ production. These results show that (i) IFN-γ production by CD8+and CD4+cells correlates with the clinical manifestations ofM. tuberculosisinfection and (ii) IFN-γ production by CD8+cells depends on CD4+cells.
Cloned Shiga Toxin 2 B Subunit Induces Apoptosis in Ramos Burkitt's Lymphoma B Cells
Authors: Marcato Paola , Mulvey George , Armstrong Glen D.
Published: July 2002

The Shiga toxins (Stx1 and Stx2), produced byShigella dysenteriaetype 1 and enterohemorrhagicEscherichia coli, consist of one A subunit and five B subunits. The Stx1 and Stx2 B subunits form a pentameric structure that binds to globotriaosylceramide (Gb3-Cer) receptors on eukaryotic cells and promotes endocytosis. The A subunit then inhibits protein biosynthesis, which triggers apoptosis in the affected cell. In addition to its Gb3-Cer binding activity, the data in the following report demonstrate that the Stx2 B pentamer induces apoptosis in Ramos Burkitt's lymphoma B cells independently of A subunit activity. Apoptosis was not observed in A subunit-free preparations of the Stx1 B pentamer which competitively inhibited Stx2 B pentamer-mediated apoptosis. The pancaspase inhibitor, Z-VAD-fmk, prevented apoptosis in Ramos cells exposed to the Stx2 B subunit, Stx1 or Stx2. Brefeldin A, an inhibitor of the Golgi transport system, also prevented Stx2 B subunit-mediated apoptosis. These observations suggest that the Stx2 B subunit must be internalized, via Gb3-Cer receptors, to induce Ramos cell apoptosis. Moreover, unlike the two holotoxins, Stx2 B subunit-mediated apoptosis does not involve inhibition of protein biosynthesis. This study provides further insight into the pathogenic potential of this family of potent bacterial exotoxins.
Role of Osteopontin in Murine Lyme Arthritis and Host Defense againstBorrelia burgdorferi
Authors: Potter Melissa R. , Rittling Susan R. , Denhardt David T. , Roper Randall J. , Weis John H. , Teuscher Cory , Weis Janis J.
Published: July 2002

Several genetic loci in the mouse have been identified that regulate the severity of Lyme arthritis. The region of chromosome 5 including the osteopontin (OPN) gene (Opn) has been identified in intercross populations of C3H/HeN × C57BL/6 and C3H/HeJ × BALB/cAnN mice. OPN is of particular interest as it is involved in the maintenance and remodeling of tissue during inflammation, it regulates production of interleukin-10 (IL-10) and IL-12 (cytokines implicated in Lyme arthritis), it is necessary for host control of certain bacterial infections, and mice displaying different severities of Lyme arthritis possess different alleles of the OPN gene. Macrophages and splenocytes from OPN-deficient mice on mixed C57BL/6J-129S or inbred 129S backgrounds were stimulated with the Pam3Cys modified lipoprotein fromBorrelia burgdorferi, OspA. OPN was not required for OspA-induced cytokine production; however, macrophages from 129S-Opn−/−mice displayed a reduced level of IL-10 production. OPN was also not required for resistance to severe arthritis, asB. burgdorferi-infected 129S-Opn−/−mice developed mild arthritis, as did their wild-type littermates. Arthritis was more severe in OPN-deficient mice on the mixed C57BL/6J-129S backgrounds than in inbred mice of either strain. This increase was most likely due to a gene(s) closely linked toOpnon chromosome 5 in conjunction with other randomly assorting genes. Deficiency in OPN did not influence the numbers of spirochetes in tissues fromB. burgdorferi-infected mice, indicating OPN is not part of the host defense to this pathogen. Interestingly, there was no alteration in theB. burgdorferi-specific antibody isotypes in OPN-deficient mice, indicating that its effect on helper T-cell responses is not relevant to the host response toB. burgdorferi.
Identification of Motility and Autoagglutination
Campylobacter jejuni
Mutants by Random Transposon Mutagenesis
Authors: Golden Neal J. , Acheson David W. K.
Published: July 2002

Campylobacter jejuni
has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of
C. jejuni
transformants. A total of 1,065
C. jejuni
transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.
EnterohemorrhagicEscherichia coliInfection Induces Interleukin-8 Production via Activation of Mitogen-Activated Protein Kinases and the Transcription Factors NF-κB and AP-1 in T84 Cells
Authors: Dahan Stephanie , Busuttil Valere , Imbert Veronique , Peyron Jean-Francois , Rampal Patrick , Czerucka Dorota
Published: July 2002

EnterohemorrhagicEscherichia coli(EHEC) infections are associated with hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). In vivo, elevated plasma levels of the proinflammatory cytokine interleukin-8 (IL-8) in EHEC-infected children are correlated with a high risk of developing HUS. As IL-8 gene transcription is regulated by the transcription factors NF-κB and AP-1, we analyzed the role of these factors in the regulation of IL-8 production after infection of the epithelial intestinal T84 cell line by EHEC. By 6 h of infection, EHEC had induced significant secretion of IL-8 (35.84 ± 6.76 ng/ml versus 0.44 ± 0.04 ng/ml in control cells). EHEC induced AP-1 and NF-κB activation by 3 h of infection. Moreover, the three mitogen-activated protein kinases (MAPK) (ERK1/2, p38, and JNK) were phosphorylated in EHEC-infected T84 cells concomitant with induction of AP-1 DNA binding activity, and IκB-α was phosphorylated and then degraded concomitant with induction of NF-κB DNA binding activity. Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and/or the proteasome inhibitor ALLN led to inhibition of the IL-8 secretion induced in EHEC-infected T84 cells. These findings demonstrate that (i) EHEC can induce in vitro a potent proinflammatory response by secretion of IL-8 and (ii) the secretion of IL-8 is due to the involvement of MAPK, AP-1, and NF-κB signaling pathways.
Protection of Killer Antiidiotypic Antibodies against Early Invasive Aspergillosis in a Murine Model of Allogeneic T-Cell-Depleted Bone Marrow Transplantation
Authors: Cenci Elio , Mencacci Antonella , Spreca Antonio , Montagnoli Claudia , Bacci Angela , Perruccio Katia , Velardi Andrea , Magliani Walter , Conti Stefania , Polonelli Luciano , Romani Luigina
Published: July 2002

Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs againstAspergillus fumigatuswas investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinatedAspergillusconidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy inA. fumigatusinfections.
Complete Nucleotide Sequence of Tn
10
Authors: Chalmers Ronald , Sewitz Sven , Lipkow Karen , Crellin Paul
Published: July 2002

The complete nucleotide sequence of Tn
10
has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn
10
constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in
Bacillus subtilis
. The glutamate permease was fully functional when expressed, but Tn
10
did not protect
Escherichia coli
from the toxic effects of various metals.
Identification of
Sinorhizobium meliloti
Genes Regulated during Symbiosis
Authors: Cabanes Didier , Boistard Pierre , Batut Jacques
Published: July 2002

RNA fingerprinting by arbitrarily primed PCR was used to isolate
Sinorhizobium meliloti
genes regulated during the symbiotic interaction with alfalfa (
Medicago sativa
). Sixteen partial cDNAs were isolated whose corresponding genes were differentially expressed between symbiotic and free-living conditions. Thirteen sequences corresponded to genes up-regulated during symbiosis, whereas three were instead repressed during establishment of the symbiotic interaction. Seven cDNAs corresponded to known or predicted
nif
and
fix
genes. Four presented high sequence similarity with genes not yet identified in
S. meliloti
, including genes encoding a component of the pyruvate dehydrogenase complex, a cell surface protein component, a copper transporter, and an argininosuccinate lyase. Finally, five cDNAs did not exhibit any similarity with sequences present in databases. A detailed expression analysis of the nine non-
nif-fix
genes provided evidence for an unexpected variety of regulatory patterns, most of which have not been described so far.
Analysis of the Polar Flagellar Gene System of
Vibrio parahaemolyticus
Authors: Kim Yun-Kyeong , McCarter Linda L.
Published: July 2002

Vibrio parahaemolyticus
has dual flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum propels the swimmer cell in liquid environments. Numerous unsheathed lateral flagella move the swarmer cell over surfaces. The polar flagellum is produced continuously, whereas the synthesis of lateral flagella is induced under conditions that impede the function of the polar flagellum, e.g., in viscous environments or on surfaces. Thus, the organism possesses two large gene networks that orchestrate polar and lateral flagellar gene expression and assembly. In addition, the polar flagellum functions as a mechanosensor controlling lateral gene expression. In order to gain insight into the genetic circuitry controlling motility and surface sensing, we have sought to define the polar flagellar gene system. The hierarchy of regulation appears to be different from the polar system of
Caulobacter crescentus
or the peritrichous system of enteric bacteria but is pertinent to many
Vibrio
and
Pseudomonas
species. The gene identity and organization of 60 potential flagellar and chemotaxis genes are described. Conserved sequences are defined for two classes of polar flagellar promoters. Phenotypic and genotypic analysis of mutant strains with defects in swimming motility coupled with primer extension analysis of flagellar and chemotaxis transcription provides insight into the polar flagellar organelle, its assembly, and regulation of gene expression.
Global Adaptations Resulting from High Population Densities in
Escherichia coli
Cultures
Authors: Liu XueQiao , Ng Christina , Ferenci Thomas
Published: July 2002

The scope of population density effects was investigated in steady-state continuous cultures of
Escherichia coli
in the absence of complications caused by transient environmental conditions and growth rates. Four distinct bacterial properties reflecting major regulatory and physiological circuits were analyzed. The metabolome profile of bacteria growing at high density contained major differences from low-density cultures. The 10-fold-elevated level of trehalose at higher densities pointed to the increased role of the RpoS sigma factor, which controls trehalose synthesis genes as well as the general stress response. There was an eightfold difference in RpoS levels between bacteria grown at 10
8
and at 10
9
cells/ml. In contrast, the cellular content of the DNA binding protein H-NS, controlling many genes in concert with RpoS, was decreased by high density. Since H-NS and RpoS also influence porin gene expression, the influence of population density on the intricate regulation of outer membrane composition was also investigated. High culture densities were found to strongly repress
ompF
porin transcription, with a sharp threshold at a density of 4.4 × 10
8
cells/ml, while increasing the proportion of OmpC in the outer membrane. The density-dependent regulation of
ompF
was maintained in
rpoS
or
hns
mutants and so was independent of these regulators. The consistently dramatic changes indicate that actively growing, high-density cultures are at least as differentiated from low-density cultures as are exponential- from stationary-phase bacteria.
AnkB, a Periplasmic Ankyrin-Like Protein in
Pseudomonas aeruginosa
, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide
Authors: Howell Michael L. , Alsabbagh Eyad , Ma Ju-Fang , Ochsner Urs A. , Klotz Martin G. , Beveridge Terry J. , Blumenthal Kenneth M. , Niederhoffer Eric C. , Morris Randall E. , Needham David , Dean Gary E. , Wani Maqsood A. , Hassett Daniel J.
Published: July 2002

In this study, we have cloned the
ankB
gene, encoding an ankyrin-like protein in
Pseudomonas aeruginosa
. The
ankB
gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location of
ankB
is 57 bp downstream of
katB
, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ∼65% α-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that
katB
and
ankB
are part of a small operon whose transcription is induced dramatically by H
2
O
2
, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an
ankB
mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H
2
O
2
, phenotypes that could be complemented in
trans
by a plasmid constitutively expressing
ankB
. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H
2
O
2
detoxification.
An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (ς
AdsA
) That Is Essential for Morphological Development in
Streptomyces griseus
Authors: Yamazaki Haruka , Ohnishi Yasuo , Horinouchi Sueharu
Published: July 2002

A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in
Streptomyces griseus
. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the
adpA
promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function ς factor belonging to a subgroup of the primary ς
70
family. The cloned gene was named AdpA-dependent sigma factor gene (
adsA
), and the gene product was named ς
AdsA
. Transcription of
adsA
depended on A-factor and AdpA, since
adsA
was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an
adpA
-disrupted strain. Consistent with this, transcription of
adsA
was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of
adsA
in some unknown way. Disruption of the chromosomal
adsA
gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that ς
AdsA
is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the
Streptomyces
species examined by Southern hybridization suggests a common role in morphogenesis in this genus.
Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation
Authors: Fujimoto David F. , Brunskill Eric W. , Bayles Kenneth W.
Published: July 2002

Penicillin-induced killing and murein hydrolase activity inStaphylococcus aureusare dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designatedlrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation oflrgABexpression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulatelrgABexpression. A mutation in theagrlocus reducedlrgABexpression approximately sixfold, while thesarmutation reducedlrgABexpression to undetectable levels.cis-acting regulatory elements involved inlrgABexpression were identified by fusing various fragments of thelrgABpromoter region to thexylEreporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximallrgABexpression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reducedlrgABpromoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate thatlrgABexpression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.
A Role for the
umuDC
Gene Products of
Escherichia coli
in Increasing Resistance to DNA Damage in Stationary Phase by Inhibiting the Transition to Exponential Growth
Authors: Murli Sumati , Opperman Timothy , Smith Bradley T. , Walker Graham C.
Published: July 2002

The
umuDC
gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis. We have recently presented evidence that supports a role for the
umuDC
gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint. Our further characterization of the growth inhibition at 30°C associated with constitutive expression of the
umuDC
gene products from a multicopy plasmid has shown that the
umuDC
gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30°C and that this is correlated with a rapid inhibition of DNA synthesis. These observations led to the finding that physiologically relevant levels of the
umuDC
gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in
E. coli
cells that have experienced DNA damage while in stationary phase. This activity of the
umuDC
gene products is correlated with an increase in survival after UV irradiation. In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity. The
umuDC
-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage. Thus, the
umuDC
gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells.
Succinyl Coenzyme A Synthetase of
Pseudomonas aeruginosa
with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase
Authors: Kapatral Vinayak , Bina Xiaowen , Chakrabarty A. M.
Published: July 2002

Pseudomonas aeruginosa
secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the
algR2
gene of mucoid
P. aeruginosa
is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the
sucCD
operon encoding the α and β subunits of Scs from
P. aeruginosa
and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from
P. aeruginosa
is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P
i
), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of
P. aeruginosa
, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid
P. aeruginosa
.
Genetic and Biochemical Characterization of a Novel
umuD
Mutation: Insights into a Mechanism for UmuD Self-Cleavage
Authors: Sutton Mark D. , Kim Melanie , Walker Graham C.
Published: July 2002

Most translesion DNA synthesis (TLS) in
Escherichia coli
is dependent upon the products of the
umuDC
genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA–single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD′. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD′ with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD′ homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD′ proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD
2
and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD
2
for self-cleavage.
Identification and Functional Characterization of Arylamine
N
-Acetyltransferases in Eubacteria: Evidence for Highly Selective Acetylation of 5-Aminosalicylic Acid
Authors: Deloménie Claudine , Fouix Sylvaine , Longuemaux Sandrine , Brahimi Naı̈ma , Bizet Chantal , Picard Bertrand , Denamur Erick , Dupret Jean-Marie
Published: July 2002

Arylamine
N
-acetyltransferase activity has been described in various bacterial species. Bacterial
N
-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time
N
-acetyltransferase activity in 11 species of
Proteobacteriaceae
from seven genera:
Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides
, and
Vibrio cholerae
. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast,
para
-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied,
Pseudomonas aeruginosa
was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a
Salmonella enterica
serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.
Complete Nucleotide Sequence and Organization of the Atrazine Catabolic Plasmid pADP-1 from
Pseudomonas
sp
.
Strain ADP
Authors: Martinez Betsy , Tomkins Jeffrey , Wackett Lawrence P. , Wing Rod , Sadowsky Michael J.
Published: July 2002

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from
Pseudomonas
sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of
s
-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPβ plasmid previously isolated from
Enterobacter aerogenes
. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn
5053
. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS
1071
and TnpA from
Pseudomonas pseudoalcaligenes
were identified and flank each of the
atzA, atzB,
and
atzC
genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes,
atzD
,
atzE
, and
atzF,
which participate in atrazine catabolism. Crude extracts from
Escherichia coli
expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from
Pseudomonas
sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid,
atzE
and
atzF
, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator.
E. coli
strains bearing
atzE
and
atzF
were shown to encode a biuret hydrolase and allophanate hydrolase, respectively.
atzDEF
are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.
RNA Polymerase Sigma Factor That Blocks Morphological Differentiation by
Streptomyces coelicolor
Authors: Gehring Amy M. , Yoo Narie J. , Losick Richard
Published: July 2002

The filamentous bacterium
Streptomyces coelicolor
undergoes a complicated process of morphological differentiation that begins with the formation of an aerial mycelium and culminates in sporulation. Genes required for the initiation of aerial mycelium formation have been termed
bld
(bald), describing the smooth, undifferentiated colonies of mutant strains. By using an insertional mutagenesis protocol that relies on in vitro transposition, we have isolated a
bld
mutant harboring an insertion in a previously uncharacterized gene,
SCE59.12c
, renamed here
rsuA
. The insertion mutant exhibited no measurable growth defect but failed to produce an aerial mycelium and showed a significant delay in the production of the polyketide antibiotic actinorhodin. The
rsuA
gene encodes an apparent anti-sigma factor and is located immediately downstream of
SCE59.13c
, renamed here
sigU
, whose product is inferred to be a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors. The absence of
rsuA
in a strain that contained
sigU
caused a block in development, and the overexpression of
sigU
in an otherwise wild-type strain caused a delay in aerial mycelium formation. However, a strain in which both
rsuA
and
sigU
had been deleted was able to undergo morphological differentiation normally. We conclude that the
rsuA
-encoded anti-sigma factor is responsible for antagonizing the function of the sigma factor encoded by
sigU
. We also conclude that the
sigU
-encoded sigma factor is not normally required for development but that its uncontrolled activity obstructs morphological differentiation at an early stage.
The
rlmB
Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation in
Escherichia coli
Authors: Lövgren J. Mattias , Wikström P. Mikael
Published: July 2002

In
Saccharomyces cerevisiae
, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposed
Escherichia coli
ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a Δ
rlmB
mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in
E. coli
.
Natural Genetic Transformation of
Streptococcus mutans
Growing in Biofilms
Authors: Li Yung-Hua , Lau Peter C. Y. , Lee Janet H. , Ellen Richard P. , Cvitkovitch Dennis G.
Published: July 2002

Streptococcus mutans
is a bacterium that has evolved to be dependent upon a biofilm “lifestyle” for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in
S. mutans
and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes,
comC
,
comD
, and
comE
, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of
comC
and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown
S. mutans
cells were transformed at a rate 10- to 600-fold higher than planktonic
S. mutans
cells. Donor DNA included a suicide plasmid,
S. mutans
chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in
S. mutans
and that the system functions optimally when the cells are living in actively growing biofilms.
Identification of an
Actinobacillus pleuropneumoniae
Consensus Promoter Structure
Authors: Doree Scott M. , Mulks Martha H.
Published: July 2002

Actinobacillus pleuropneumoniae
promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless
luxAB
genes of
Vibrio harveyi
. The degree of expression was quantifiable, and only high-expression or “hot” promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for
A. pleuropneumoniae
was identified. The consensus promoter sequence for
A. pleuropneumoniae
was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the
A. pleuropneumoniae
consensus with other prokaryotic consensus promoters showed that the
A. pleuropneumoniae
consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical −10 element and a similar but truncated −35 element. However, the
A. pleuropneumoniae
consensus promoter is unique in the spacing between the −10 and −35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an
A. pleuropneumoniae
promoter is shorter than the spacing identified for
Escherichia coli
and
Bacillus subtilis
promoters.
Construction and Characterization of Mutations at Codon 751 of the
Escherichia coli gyrB
Gene That Confer Resistance to the Antimicrobial Peptide Microcin B17 and Alter the Activity of DNA Gyrase
Authors: del Castillo Francisco J. , del Castillo Ignacio , Moreno Felipe
Published: July 2002

Microcin B17 is a peptide antibiotic that inhibits DNA replication in
Escherichia coli
by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same
gyrB
point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.
Characterization of a
Mycoplasma pneumoniae hmw3
Mutant: Implications for Attachment Organelle Assembly
Authors: Willby Melisa J. , Krause Duncan C.
Published: July 2002

The proteins required for adherence of the pathogen
Mycoplasma pneumoniae
to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.
Global Transcriptional Analysis ofclpPMutations of Type 2Streptococcus pneumoniaeand Their Effects on Physiology and Virulence
Authors: Robertson Gregory T. , Ng Wai-Leung , Foley Joseph , Gilmour Raymond , Winkler Malcolm E.
Published: July 2002

Streptococcus pneumoniaeis an important human pathogen that contains single copies of genes encoding the ClpP and FtsH ATP-dependent proteases but lacks the Lon and HslV proteases. We constructed and characterized the phenotypes ofclpP,clpC, andclpXdeletion replacement mutants, which lack the ClpP protease subunit or the putative ClpC or ClpX ATPase specificity factor. A ΔclpPmutant, but not a ΔclpCor ΔclpXmutant, of the virulent D39 type 2 strain ofS. pneumoniaegrew poorly at 30°C and failed to grow at 40°C. Despite this temperature sensitivity, transcription of the heat shock regulon determined by microarray analysis was induced in a ΔclpPmutant, which was also more sensitive to oxidative stress by H2O2and to puromycin than itsclpP+parent strain. A ΔclpPmutant, but not a ΔclpCmutant, was strongly attenuated for virulence in the murine lung and sepsis infection models. All of these phenotypes were complemented in a ΔclpP/clpP+merodiploid strain. Consistent with these complementation patterns,clpPwas found to be in a monocistronic operon, whose transcription was induced about fivefold by heat shock inS. pneumoniaeas determined by Northern and real-time reverse transcription-PCR analyses. BesidesclpP, transcription ofclpC,clpE, andclpL, but notclpXorftsH, was induced by heat shock or entry into late exponential growth phase. Microarray analysis of ΔclpPmutants showed a limited change in transcription pattern (≈80 genes) consistent with these phenotypes, including repression of genes involved in oxidative stress, metal ion transport, and virulence. In addition, transcription of the early and late competence regulon was induced in the ΔclpPmutant, and competence gene expression and DNA uptake seemed to be constitutively induced throughout growth. Together, these results indicate that ClpP-mediated proteolysis plays a complex and central role in numerous pneumococcal stress responses, development of competence, and virulence.
ZipA Is Required for Recruitment of FtsK, FtsQ, FtsL, and FtsN to the Septal Ring in
Escherichia coli
Authors: Hale Cynthia A. , de Boer Piet A. J.
Published: July 2002

The septal ring in
Escherichia coli
consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.