bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Switch On of
ssgA
by A-Factor, Which Is Essential for Spore Septum Formation in
Streptomyces griseus
Authors: Yamazaki Haruka , Ohnishi Yasuo , Horinouchi Sueharu 
Published: January 2003

ABSTRACT

A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone) triggers morphological development and secondary metabolism in
Streptomyces griseus
. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of
ssgA
, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about −235 (site 1), −110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of
ssgA. ssgA
had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of
ssgA
. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of
ssgA
required the extracytoplasmic function sigma factor, σ
AdsA
, in addition to AdpA. However, it was unlikely that σ
AdsA
recognized the two
ssgA
promoters, since their −35 and −10 sequences were not similar to the promoter sequence motifs recognized by σ
BldN
, a σ
AdsA
homologue of
Streptomyces coelicolor
A3(2). An
ssgA
disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that
ssgA
is a member of the
whi
genes. Transcriptional analysis of
ssfR
, located just upstream of
ssgA
and encoding an IclR-type transcriptional regulator, suggested that no read-through from
ssfR
into
ssgA
occurred, and
ssgA
was transcribed in the absence of
ssfR. ssgA
was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an
ssfR
disruptant also showed a
whi
phenotype.
The Small Subunit of M ·
Aqu
I Is Responsible for Sequence-Specific DNA Recognition and Binding in the Absence of the Catalytic Domain
Authors: Pinarbasi Hatice , Pinarbasi Ergun , Hornby David P. 
Published: January 2003

ABSTRACT

Aqu
I DNA methyltransferase (M ·
Aqu
I) catalyzes the transfer of a methyl group from
S
-adenosyl-
l
-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5′-CCCGGG-3′. M ·
Aqu
I is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M ·
Hha
I. Recently, we reported the subcloning, overexpression, and purification of the subunits (α and β) of M ·
Aqu
I separately. Here we describe the DNA binding properties of M ·
Aqu
I. The results presented here indicate that the β subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M ·
Aqu
I involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the β subunit. The α subunit enhances the binding of the β subunit to DNA specifically and nonspecifically. It is likely that the addition of the α subunit to the β subunit stabilizes the conformation of the β subunit and thereby enhances its affinity for DNA indirectly. Addition of
S
-adenosyl-
l
-methionine and its analogues
S
-adenosyl-
l
-homocysteine and sinefungin enhances binding, but only in the presence of the α subunit. These compounds did not have any effect on DNA binding by the β subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the β subunit alone did not form a covalent complex with its specific sequence in the absence or presence of
S
-adenosyl-
l
-methionine. However, the addition of the α subunit to the β subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.
KtrAB and KtrCD: Two K+Uptake Systems inBacillus subtilisand Their Role in Adaptation to Hypertonicity
Authors: Holtmann Gudrun , Bakker Evert P. , Uozumi Nobuyuki , Bremer Erhard 
Published: January 2003

ABSTRACTRecently, a new type of K+transporter, Ktr, has been identified in the bacteriumVibrio alginolyticus(T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol.180:3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence ofBacillus subtiliscontains two genes for each of these subunits:yuaA(ktrA) andykqB(ktrC) encode homologues to theV. alginolyticusKtrA protein, andyubG(ktrB) andykrM(ktrD) encode homologues to theV. alginolyticusKtrB protein. We constructed gene disruption mutations in each of the fourB. subtilis ktrgenes and used this isogenic set of mutants for K+uptake experiments. Preliminary K+transport assays revealed that the KtrAB system has a moderate affinity with aKmvalue of approximately 1 mM for K+, while KtrCD has a low affinity with aKmvalue of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K+uptake activity, demonstrating that KtrAB and KtrCD systems are the major K+transporters ofB. subtilis. Northern blot analyses revealed thatktrAandktrBare cotranscribed as an operon, whereasktrCandktrD, which occupy different locations on theB. subtilischromosome, are expressed as single transcriptional units. The amount of K+in the environment or the salinity of the growth medium did not influence the amounts of the variousktrtranscripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In thektrABstrain, the osmotically mediated growth defect was associated with a rapid loss of K+ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically inducedproHJ,opuA, andgsiBgenes was not impaired, demonstrating that a high cytoplasmic K+concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K+uptake via KtrAB and KtrCD is an important facet in the cellular defense ofB. subtilisagainst both suddenly imposed and prolonged osmotic stress.
Genetic and Biochemical Analysis of Phosphatase Activity of
Escherichia coli
NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein
Authors: Pioszak Augen A. , Ninfa Alexander J. 
Published: January 2003

ABSTRACT

Mutant forms of
Escherichia coli
NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the
glnL (ntrB)
gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the “back” or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the “back” of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.
Whole-Genome Sequence Variation among Multiple Isolates of
Pseudomonas aeruginosa
Authors: Spencer David H. , Kas Arnold , Smith Eric E. , Raymond Christopher K. , Sims Elizabeth H. , Hastings Michele , Burns Jane L. , Kaul Rajinder , Olson Maynard V. 
Published: January 2003

ABSTRACT

Whole-genome shotgun sequencing was used to study the sequence variation of three
Pseudomonas aeruginosa
isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, ∼10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core
P. aeruginosa
backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.
Effects of the Chromosome Partitioning Protein Spo0J (ParB) onoriCPositioning and Replication Initiation inBacillus subtilis
Authors: Lee Philina S. , Lin Daniel Chi-Hong , Moriya Shigeki , Grossman Alan D. 
Published: January 2003

ABSTRACTSpo0J (ParB) ofBacillus subtilisis a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomaloriCregion, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In aspo0Jnull mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parSsites) were inserted at various chromosomal locations in the absence of six of the eight knownparSsites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomalparSsites to the cell quarters.spo0Jalso affected chromosome positioning during sporulation. Aspo0Jnull mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, aspo0Jnull mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays oflacoperators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that thespo0Jmutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.
Growth Phase-Coupled Alterations in Cell Structure and Function of
Escherichia coli
Authors: Makinoshima Hideki , Aizawa Shin-Ichi , Hayashi Hideo , Miki Takeyoshi , Nishimura Akiko , Ishihama Akira 
Published: January 2003

ABSTRACT

Escherichia coli
cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant
E. coli
with the disrupted
rpoS
gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type
E. coli
, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase
E. coli
cells.
Glycerol-3-Phosphate Acquisition in Spirochetes: Distribution and Biological Activity of Glycerophosphodiester Phosphodiesterase (GlpQ) amongBorreliaSpecies
Authors: Schwan Tom G. , Battisti James M. , Porcella Stephen F. , Raffel Sandra J. , Schrumpf Merry E. , Fischer Elizabeth R. , Carroll James A. , Stewart Philip E. , Rosa Patricia , Somerville Greg A. 
Published: January 2003

ABSTRACTRelapsing-fever spirochetes achieve high cell densities (>108/ml) in their host's blood, while Lyme disease spirochetes do not (<105/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood.Borrelia hermsii,a tick-borne relapsing-fever spirochete, contains orthologs toglpQandglpT,genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstratedglpQandglpTin all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete,Borrelia burgdorferi,that was transformed with a shuttle vector containingglpTQfromB. hermsiiproduced active enzyme, which demonstrated the association ofglpQwith the hydrolysis of phospholipids. Sequence analysis ofB. hermsiiidentifiedglpF, glpK,andglpA,which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present inB. burgdorferi.All spirochetes examined hadgpsA,which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.
Detection of Other Microbial Species bySalmonella: Expression of the SdiA Regulon
Authors: Smith Jenée N. , Ahmer Brian M. M. 
Published: January 2003

ABSTRACTSalmonella,Escherichia, andKlebsiellado not encode any recognized type ofN-acylhomoserine lactone (AHL) synthase, and consistent with this, they do not synthesize AHLs under any conditions tested. However, they do encode an AHL receptor of the LuxR family, named SdiA. MudJ fusions in four loci are known to respond to plasmid-encodedsdiAinSalmonella, but only thercklocus has been described. Here we report the location and sequence analysis of the remaining three loci. Thesrg-6::MudJ is withingtgAof the gifsy-2 prophage, and thesrg-7::MudJ is within PSLT61 of the virulence plasmid. Both fusions are in the antisense orientation. The third fusion,srgE5::MudJ, is within a horizontally acquired gene of unknown function at 33.6 centisomes that we have namedsrgE. Previously,sdiAexpressed from its natural position in the chromosome was demonstrated to activate a plasmid-based transcriptional fusion to therckpromoter in response to AHL production by other bacterial species. However, the MudJ fusions did not respond to chromosomalsdiA. Here we report that MudJ fusions to three of the four loci (notsrg-6) are activated by AHL in ansdiA-dependent manner during growth in motility agar (0.25% agar) but not during growth in top agar (0.7% agar) or on agar plates (1.2% agar). In motility agar, thesrgEpromoter responds tosdiAat 30°C and higher while therckandsrg-7promoters respond only at 37 or 42°C. Substantial AHL-independent SdiA activity was observed at 30°C but not at 37°C.
Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein
Authors: Maestro Beatriz , Sanz Jesús M. , Díaz-Orejas Ramón , Fernández-Tresguerres Elena 
Published: January 2003

ABSTRACT

We report here the isolation and analysis of novel
repA
host range mutants of pPS10, a plasmid originally found in
Pseudomonas savastanoi
. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in
Escherichia coli
at 37°C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in
Pseudomonas
, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than
E
.
coli
such as
Pseudomonas putida
and
Alcaligenes faecalis
. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different
repA
mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA.
Role of Two Novel Two-Component Regulatory Systems in Development and Phosphatase Expression in
Myxococcus xanthus
Authors: Moraleda-Muñoz Aurelio , Carrero-Lérida Juana , Pérez Juana , Muñoz-Dorado José 
Published: January 2003

ABSTRACT

We have cloned a two-component regulatory system (
phoR2-phoP2
) of
Myxococcus xanthus
while searching for genes that encode proteins with phosphatase activity, where
phoR2
encodes the histidine kinase and
phoP2
encodes the response regulator. A second system,
phoR3-phoP3
, was identified and isolated by using
phoP2
as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the
pph1
gene.
Identification and Characterization of aVibrio choleraeGene,mbaA, Involved in Maintenance of Biofilm Architecture
Authors: Bomchil Natalia , Watnick Paula , Kolter Roberto 
Published: January 2003

ABSTRACTThe formation of biofilms is thought to play a key role in the environmental survival of the marine bacteriumVibrio cholerae. Although the factors involved inV.choleraeattachment to abiotic surfaces have been extensively studied, relatively little is known about the mechanisms involved in the subsequent maturation of the biofilms. Here we report the identification of a novel gene, which we have namedmbaA(for maintenance of biofilm architecture), that plays a role in the formation and maintenance of the highly organized three-dimensional architecture ofV.choleraeEl Tor biofilms. We demonstrate that although the absence ofmbaAdoes not significantly affect the initial attachment of cells onto the surface, it leads to the formation of biofilms that lack the typical structure, including the pillars of cells separated by fluid-filled channels that are evident in mature wild-type biofilms. Microscopic analysis indicates that the absence ofmbaAleads to an increase in the amount of extracellular matrix material in the biofilms. The predictedmbaAproduct is a member of a family of regulatory proteins, containing GGDEF and EAL domains, suggesting that MbaA regulates the synthesis of some component of the biofilm matrix.
Subcellular Localization of a Small Sporulation Protein inBacillus subtilis
Authors: van Ooij Christiaan , Losick Richard 
Published: January 2003

ABSTRACTSpoVM is an unusually small (26-residue-long) protein that is produced in the mother cell chamber of the sporangium during the process of sporulation inBacillus subtilis. We investigated the subcellular localization of SpoVM, which is believed to be an amphipathic α-helix, by using a fusion of the sporulation protein to the green fluorescence protein (GFP). We found that SpoVM-GFP is recruited to the polar septum shortly after the sporangium undergoes asymmetric division and that the fusion protein localizes to the mother cell membrane that surrounds the forespore during the subsequent process of engulfment. We identified a patch of three residues near the N terminus of the proposed α-helix that is needed both for proper subcellular localization and for SpoVM function. We also identified a patch of residues on the opposite face of the helix and residues near both ends of the protein that are needed for SpoVM function but not for subcellular localization. Subcellular localization of SpoVM-GFP was found to require an unknown gene(s) under the control of the mother cell transcription factor σE. We propose that the N-terminal patch binds to an unknown anchoring protein that is produced under the control of σEand that other residues important in SpoVM function to recruit an unknown sporulation protein(s) to the mother cell membrane that surrounds the forespore. Our results provide evidence that SpoVM function depends on proper subcellular localization.