bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of
Xanthobacter flavus
Authors: van Keulen Geertje , Ridder Anja N. J. A. , Dijkhuizen Lubbert , Meijer Wim G.
Published: January 2003

The LysR-type transcriptional regulator CbbR controls the expression of the
cbb
and
gap-pgk
operons in
Xanthobacter flavus
, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO
2
fixation. The
cbb
operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and
cbb
promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR
1
is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the
cbb
operon promoter. In addition, it is required for negative autoregulation of
cbbR
expression. IR
3
represents the main low-affinity binding site of CbbR. Binding to IR
3
occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR
1
also prevent binding to the low-affinity site. Although mutations in IR
3
have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR
3
partially overlaps with the −35 region of the
cbb
operon promoter. Mutations in IR
2
do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the
cbb
operon promoter. This IR
2
binding site is therefore critical for transcriptional activation by CbbR.
Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites
Authors: Vaillancourt Frédéric H. , Haro María-Amparo , Drouin Nathalie M. , Karim Zamil , Maaroufi Halim , Eltis Lindsay D.
Published: January 2003

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from
Burkholderia
sp. strain LB400 (DHBD
LB400
; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-dichloro-2,3-dihydroxybiphenyl (2′,6′-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBD
P6
-I and DHBD
P6
-III, two evolutionarily divergent isozymes from
Rhodococcus globerulus
strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBD
LB400
, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBD
P6
-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBD
P6
-I were among the best for DHBD
P6
-III (4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB; DHBD
P6
-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBD
P6
-I and 2′-Cl DHB for DHBD
P6
-III. As observed in DHBD
LB400
, chloro substituents influenced the reactivity of the dioxygenases with O
2
. For example, the apparent specificities of DHBD
P6
-I and DHBD
P6
-III for O
2
in the presence of 2′-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBD
P6
-I and DHBD
P6
-III shared the relative inability of DHBD
LB400
to cleave 2′,6′-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s
−1
, respectively). However, these isozymes had remarkably different apparent
K
m
values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 μM for DHBD
LB400
, DHBD
P6
-I, and DHBD
P6
-III, respectively). The markedly different reactivities of DHBD
P6
-I and DHBD
P6
-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of
R. globerulus
P6.
Pyoverdine-Mediated Regulation of FpvA Synthesis in
Pseudomonas aeruginosa
: Involvement of a Probable Extracytoplasmic-Function Sigma Factor, FpvI
Authors: Rédly Gyula Alan , Poole Keith
Published: January 2003

A search of the
pvd
pyoverdine biosynthesis locus of
Pseudomonas aeruginosa
identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in
Escherichia coli
and
Pseudomonas putida
. Deletion of this gene, dubbed
fpvI
, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and
fpvA
gene expression, while the cloned gene stimulated
fpvA
expression. A Fur-binding site was identified immediately upstream of
fpvI
, consistent with the observed iron-regulated expression of
fpvI
and
fpvA
.
Molecular Characteristics of Spontaneous Deletions in the Hyperthermophilic Archaeon
Sulfolobus acidocaldarius
Authors: Grogan Dennis W. , Hansen Josh E.
Published: January 2003

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of
Sulfolobus acidocaldarius
for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous
pyrE
mutations, corresponding to a frequency of about 10
−8
per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the
pyrE
gene and that duplication mutations in
pyrE
reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in
pyrE
, and another pair of deletions shared an octanucleotide at their 3′ ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the
S
.
acidocaldarius pyrE
gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.
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

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

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

Recently, 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

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

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

Spo0J (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

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

Relapsing-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

Salmonella,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

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

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

The 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

SpoVM 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.
Biofilm Growth and Detachment of
Actinobacillus actinomycetemcomitans
Authors: Kaplan Jeffrey B. , Meyenhofer Markus F. , Fine Daniel H.
Published: January 2003

The gram-negative, oral bacterium
Actinobacillus actinomycetemcomitans
has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of
A. actinomycetemcomitans
form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of
A. actinomycetemcomitans
biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that
A. actinomycetemcomitans
developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that
A. actinomycetemcomitans
biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.
cis
Elements Necessary for Developmental Expression of a
Myxococcus xanthus
Gene That Depends on C Signaling
Authors: Viswanathan Poorna , Kroos Lee
Published: January 2003

Cell contact-mediated C signaling coordinates morphogenesis and gene expression during development of
Myxococcus xanthus
. One promoter that depends on C signaling for transcription lies upstream of Ω4403, the site of a Tn
5 lac
insertion in the genome. The Ω4403 promoter has a C-box sequence centered at −49 bp that matches the consensus 5′-CAYYCCY-3′, which is found in several C-signal-dependent promoters. Mutational analysis of the Ω4403 promoter region was performed to test the importance of the C box and to identify other
cis
-acting elements. A 6-bp change in the −10 region eliminated promoter activity, but a 6-bp change in the −35 region decreased activity only about twofold. Certain single-base-pair changes in the C box centered at −49 bp abolished promoter activity, establishing the importance of this sequence element. Single-base-pair changes in a C-box-like sequence centered at −77 bp also abolished promoter activity, but the pattern of mutational effects was different from that for the C box centered at −49 bp. Additional single-base-pair changes indicated that all 10 bp from −79 to −70 bp are important for Ω4403 promoter activity. Mutations at −59, −61, −62, and −63 bp also abolished promoter activity, defining a 5-bp element from −63 to −59 bp. This 5-bp element is separated from the 10-bp element (i.e., −79 to −70 bp) by 6 bp that can be changed without loss of promoter activity. Likewise, the 5 bp between the 5-bp element and the C box can be changed without loss of activity, but deletion of these 5 bp abolished activity, indicating that spacing is important. Sequences similar to the 5- and 10-bp elements, as well as the C box, are present in other C-signal-dependent promoters, suggesting some similarity in the regulatory mechanisms, but there are also indications that these
cis
elements do not function identically in the different promoters.
ldpA
Encodes an Iron-Sulfur Protein Involved in Light-Dependent Modulation of the Circadian Period in the Cyanobacterium
Synechococcus
elongatus
PCC 7942
Authors: Katayama Mitsunori , Kondo Takao , Xiong Jin , Golden Susan S.
Published: January 2003

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium
Synechococcus
elongatus
PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named
ldpA
(for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in
S
.
elongatus
, lengthening the period at low light intensities. The
ldpA
gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.
Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in
Bacillus subtilis
Authors: McPherson Derrell C. , Popham David L.
Published: January 2003

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The
Bacillus subtilis
genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.
Identification of Genes Required for Synthesis of the Adhesive Holdfast in
Caulobacter crescentus
Authors: Smith Chris S. , Hinz Aaron , Bodenmiller Diane , Larson David E. , Brun Yves V.
Published: January 2003

Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium
Caulobacter crescentus
is mediated by a polar organelle called the “holdfast,” which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of
N
-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (
hfs
) as well as in previously identified polar development genes (
podJ
and
pleC
), and the holdfast attachment genes (
hfa
). Clean deletions of three of the four genes in the
hfs
gene cluster (
hfsDAB
) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for
N
-acetylglucosamine. Transmission electron microscopy indicated that the
hfsDAB
mutants fail to synthesize a holdfast at the stalk tip. The predicted
hfs
gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from
Xanthomonas campestris
, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from
Escherichia coli
, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the
hfs
genes play an important role in holdfast export.
Proteomic Analysis of the Spore Coats of
Bacillus subtilis
and
Bacillus anthracis
Authors: Lai Erh-Min , Phadke Nikhil D. , Kachman Maureen T. , Giorno Rebecca , Vazquez Santiago , Vazquez Jenny A. , Maddock Janine R. , Driks Adam
Published: January 2003

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of
Bacillus subtilis
and
Bacillus anthracis
spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38
B
.
subtilis
spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in
B
.
anthracis
and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished
B
.
anthracis
genome for potential coat proteins. Our analysis suggests that the
B
.
subtilis
and
B
.
anthracis
coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop
B
.
anthracis
detection methods and understand the ecological role of the coat.
Differentiation and Anaerobiosis in Standing Liquid Cultures of
Streptomyces coelicolor
Authors: van Keulen Geertje , Jonkers Henk M. , Claessen Dennis , Dijkhuizen Lubbert , Wösten Han A. B.
Published: January 2003

Streptomyces coelicolor
differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.
2-Aminopurine Allows Interspecies Recombination by a Reversible Inactivation of the
Escherichia coli
Mismatch Repair System
Authors: Matic Ivan , Babic Ana , Radman Miroslav
Published: January 2003

2-Aminopurine treatment of
Escherichia coli
induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a
Salmonella enterica
serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.
Identification and Characterization of the
gerH
Operon of
Bacillus anthracis
Endospores: a Differential Role for Purine Nucleosides in Germination
Authors: Weiner Matthew A. , Read Timothy D. , Hanna Philip C.
Published: January 2003

We identified a tri-cistronic operon,
gerH
, in
Bacillus anthracis
that is important for endospore germination triggered by two distinct germination response pathways termed inosine-His and purine-Ala. Together, the two pathways allow
B
.
anthracis
endospores a broader recognition of purines and amino acids that may be important for host-mediated germination.
The Periplasmic Protein MppA Requires an Additional Mutated Locus To Repress
marA
Expression in
Escherichia coli
Authors: Bina Xiaowen , Perreten Vincent , Levy Stuart B.
Published: January 2003

Escherichia coli
strain TP985, which has an insertional mutation in the gene for the periplasmic murein tripeptide binding protein MppA, was previously reported to overproduce MarA and exhibit a multiple-antibiotic resistance (Mar) phenotype (H. Li and J. T. Park, J. Bacteriol.
181:
4842-4847, 1999). We found that TP985 contained a previously unrecognized
marR
mutation which was responsible for the Mar phenotype. Transduction of the
mppA
mutation from TP985 to another wild-type strain did not affect antibiotic susceptibility. Overproduction of MppA repressed
marA
transcription in TP985 but not in other
mppA
or
marR
mutants. Therefore, TP985 contains an additional unknown mutation(s) that facilitates the repression of
marA
expression by MppA.
Overrepresentation of a Gene Family Encoding Extracytoplasmic Solute Receptors in
Bordetella
Authors: Antoine Rudy , Jacob-Dubuisson Françoise , Drobecq Hervé , Willery Eve , Lesjean Sarah , Locht Camille
Published: January 2003

A family of genes that are likely to encode extracytoplasmic solute receptors is strongly overrepresented in several β-proteobacteria, including
Bordetella pertussis
. This gene family, of which members have been called
bug
genes, contains some examples that are contained within polycistronic operons coding for tripartite uptake transporters of the TTT family, while the vast majority are “orphan” genes. Proteomic and functional analyses demonstrated that several of these genes are expressed in
B. pertussis
, and one is involved in citrate uptake. The
bug
genes probably form an ancient family that has been subjected to a large expansion in a restricted phylogenic group.
Corrected Sequence of the Bacteriophage P22 Genome
Authors: Pedulla Marisa L. , Ford Michael E. , Karthikeyan Tharun , Houtz Jennifer M. , Hendrix Roger W. , Hatfull Graham F. , Poteete Anthony R. , Gilcrease Eddie B. , Winn-Stapley Danella A. , Casjens Sherwood R.
Published: January 2003

We report the first accurate genome sequence for bacteriophage P22, correcting a 0.14% error rate in previously determined sequences. DNA sequencing technology is now good enough that genomes of important model systems like P22 can be sequenced with essentially 100% accuracy with minimal investment of time and resources.
Prokaryotic Utilization of the Twin-Arginine Translocation Pathway: a Genomic Survey
Authors: Dilks Kieran , Rose R. Wesley , Hartmann Enno , Pohlschröder Mechthild
Published: January 2003

The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.
The requirements of the Data Protection Act 1998 for the processing of medical data
Authors: Boyd P
Published: February 2003

The Data Protection Act 1998 presents a number of significant challenges to data controllers in the health sector. To assist data controllers in understanding their obligations under the act, the Information Commissioner has published guidance,The Use and Disclosure of Health Data, which is reproduced here. The guidance deals, among other things, with the steps that must be taken to obtain patient data fairly, the implied requirements of the act to use anonymised or psuedonymised data where possible, an exemption applicable principally to records based research, the right of patients to object to the processing of their data, and the interface of the act and the common law duty of confidence.
American biofutures: ideology and utopia in the Fukuyama/Stock debate
Authors: Ashcroft R E
Published: February 2003

Francis Fukuyama, in hisOur Posthuman Future, and Gregory Stock, in hisRedesigning Humans, present competing versions of the biomedical future of human beings, and debate the merits of more or less stringent regimes of regulation for biomedical innovation. In this article, these positions are shown to depend on a shared discourse of market liberalism, which limits both the range of ends for such innovation discussed by the authors, and the scope of their policy analyses and proposals. A proper evaluation of the human significance and policy imperatives for biomedical innovation needs to be both more utopian in its imagination, and more sophisticated in its political economy. In essence, the Fukuyama/Stock debate tells us more about current US political ideology than it does about the morality of human genetic and biopsychological engineering.