bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrophoretic Mobility of
Bacillus subtilis
Knockout Mutants with and without Flagella
Authors: Okuda Shujiro , Igarashi Ryosuke , Kusui Yusuke , Kasahara Yasuhiro , Morisaki Hisao 
Published: June 2003

ABSTRACT

Mutants of
Bacillus subtilis
168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.
Repression of Phenazine Antibiotic Production in
Pseudomonas aureofaciens
Strain 30-84 by RpeA
Authors: Whistler Cheryl A. , Pierson Leland S. 
Published: June 2003

ABSTRACT

Pseudomonas aureofaciens
strain 30-84 is a biological control bacterium that utilizes a two-component GacS/GacA regulatory system interconnected with the PhzR/PhzI quorum sensing system to positively regulate biosynthesis of phenazine antibiotics that contribute to its association with plant hosts. To date, no negative regulators of phenazine production have been identified, nor has the role of repression been studied. Here we describe a novel repressor of secondary metabolism in
P. aureofaciens
strain 30-84, RpeA, whose deduced amino acid sequence is similar to those of a group of putative two-component regulatory systems of unknown function found in several animal and plant-pathogenic bacteria. In minimal medium where phenazine production is very low, inactivation of the
rpeA
gene enhanced phenazine biosynthetic gene expression and increased phenazine production but did not increase quorum sensing signal accumulation. Furthermore, RpeA functioned to block phenazine biosynthetic gene transcription in minimal medium even when quorum-sensing signals were at a level that was sufficient for induction of phenazine gene expression in rich medium. Additionally, in the absence of
rpeA
, the quorum sensor PhzR was not required for phenazine production. Although repression plays a critical role in phenazine regulation, the
rpeA
mutation could not bypass the requirement for a functional GacS/GacA system, demonstrating that activation is required even in the absence of the RpeA repressor. This study reinforces that multiple signals, including nutrition and population density, are integrated to control the appropriate expression of phenazine antibiotics.
Probing the Catalytic Activity of a Cell Division-Specific Transpeptidase In Vivo with β-Lactams
Authors: Eberhardt Christian , Kuerschner Lars , Weiss David S. 
Published: June 2003

ABSTRACT

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of
Escherichia coli
. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three β-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the
ftsA3
(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.
Molecular Analysis of Transport and Oligomerization of the
Yersinia enterocolitica
Adhesin YadA
Authors: Roggenkamp Andreas , Ackermann Nikolaus , Jacobi Christoph A. , Truelzsch Konrad , Hoffmann Harald , Heesemann Jürgen 
Published: June 2003

ABSTRACT

The
Yersinia
adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus. For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four β-strands. To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants. Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis. We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain. Thus, YadA meets all the criteria of an autotransporter. The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters. Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk. However, the YadA-binding module did not confer serum resistance. Mutants lacking the head and neck domain were resistant to complement-mediated lysis. In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model.
Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in
Escherichia coli
K-12
Authors: Enz Sabine , Brand Heidi , Orellana Claudia , Mahren Susanne , Braun Volkmar 
Published: June 2003

ABSTRACT

Transcription of the
fecABCDE
ferric citrate transport genes of
Escherichia coli
K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm. FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor. In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR
237-317
fragment interacts with the N-terminal FecA
1-79
fragment. In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis. They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins. The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium. Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E). One FecR mutant, FecR (D138E, V197A), induced
fecA
promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA. The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the
fec
transport genes and identified sites in FecR and FecA that are important for signal transduction.
Identification and Characterization of Transposable Elements of
Paracoccus pantotrophus
Authors: Bartosik Dariusz , Sochacka Marta , Baj Jadwiga 
Published: June 2003

ABSTRACT

We studied diversity and distribution of transposable elements residing in different strains (DSM 11072, DSM 11073, DSM 65, and LMD 82.5) of a soil bacterium
Paracoccus pantotrophus
(α
-Proteobacteria
). With application of a shuttle entrapment vector pMEC1, several novel insertion sequences (ISs) and transposons (Tns) have been identified. They were sequenced and subjected to detailed comparative analysis, which allowed their characterization (i.e., identification of transposase genes, terminal inverted repeats, as well as target sequences) and classification into the appropriate IS or Tn families. The frequency of transposition of these elements varied and ranged from 10
−6
to 10
−3
depending on the strain. The copy number, localization (plasmid or chromosome), and distribution of these elements in the
Paracoccus
species
P. pantotrophus
,
P. denitrificans
,
P. methylutens
,
P. solventivorans
, and
P. versutus
were analyzed. This allowed us to distinguish elements that are common in paracocci (IS
Ppa2
, IS
Ppa3
—both of the IS
5
family—and IS
Ppa5
of IS
66
family) as well as strain-specific ones (IS
Ppa1
of the IS
256
family, IS
Ppa4
of the IS
5
family, and Tn
3434
and Tn
5393
of the Tn
3
family), acquired by lateral transfer events. These elements will be of a great value in the design of new genetic tools for paracocci, since only one element (IS
1248
of
P. denitrificans
) has been described so far in this genus.
Cluster of Type IV Secretion Genes inHelicobacter pylori's Plasticity Zone
Authors: Kersulyte Dangeruta , Velapatiño Billie , Mukhopadhyay Asish K. , Cahuayme Lizbeth , Bussalleu Alejandro , Combe Juan , Gilman Robert H. , Berg Douglas E. 
Published: June 2003

ABSTRACTSome genes present in only certain strains of the genetically diverse gastric pathogenHelicobacter pylorimay affect its phenotype and/or evolutionary potential. Here we describe a new 16.3-kb segment, 7 of whose 16 open reading frames are homologs of type IV secretion genes (virB4,virB7tovirB11, andvirD4), the third such putative secretion gene cluster found inH. pylori. This segment, to be calledtfs3, was discovered by subtractive hybridization and chromosome walking. Full-length and truncatedtfs3elements were found in 20 and 19%, respectively, of 94 strains tested, which were from Spain, Peru, India, and Japan. Atfs3remnant (6 kb) was found in an archived stock of reference strain J99, although it was not included in this strain's published genome sequence. PCR and DNA sequence analyses indicated the following. (i)tfs3's ends are conserved. (ii) Right-end insertion occurred at one specific site in a chromosomal region that is varied in gene content and arrangement, the “plasticity zone.” (iii) Left-end insertion occurred at different sites in each of nine strains studied. (iv) Sequences next to the right-end target intfs3-free strains were absent from most strains carrying full-lengthtfs3elements. These patterns suggested insertion by a transposition-like event, but one in which targets are chosen with little or no specificity at the left end and high specificity at the right end, thereby deleting the intervening DNA.
Membrane Interaction of the Glycosyltransferase MurG: a Special Role for Cardiolipin
Authors: van den Brink-van der Laan Els , Boots Jan-Willem P. , Spelbrink Robin E. J. , Kool Gerda M. , Breukink Eefjan , Killian J. Antoinette , de Kruijff Ben 
Published: June 2003

ABSTRACT

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of
Escherichia coli
MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from
E. coli
in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 ± 1 to 7 ± 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 ± 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.
Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
Authors: Ducote Matthew J. , Pettis Gregg S. 
Published: June 2003

ABSTRACT

The conjugative plasmid pIJ101 of the spore-forming bacterium
Streptomyces lividans
contains a regulatory gene,
korB
, whose product is required to repress potentially lethal expression of the pIJ101
kilB
gene. The KorB protein also autoregulates
korB
gene expression and may be involved in control of pIJ101 copy number. KorB (pIJ101) is expressed as a 10-kDa protein in
S. lividans
that is immediately processed to a mature 6-kDa repressor molecule. The conjugative
Streptomyces cyanogenus
plasmid pSB24.1 is deleted upon entry into
S. lividans
to form pSB24.2, a nonconjugative derivative that contains a
korB
gene nearly identical to that of pIJ101. Previous evidence that
korB
of pSB24.2 is capable of overriding pIJ101
kilB
-associated lethality supported the notion that pIJ101 and pSB24.2 encode highly related, perhaps even identical conjugation systems. Here we show that KorB (pIJ101) and KorB (pSB24.2) repress transcription from the pIJ101
kilB
promoter equally well, although differences exist with respect to their interactions with
kilB
promoter sequences. Despite high sequence and functional similarities, KorB (pSB24.2) was found to exist as multiple stable forms ranging in size from 10 to 6 kDa both in
S. lividans
and
S. cyanogenus
. Immediate processing of KorB (pIJ101) exclusively to the 6-kDa repressor form meanwhile was conserved between the two species. A feature common to both proteins was a marked increase in expression or accumulation upon sporulation, an occurrence that may indicate a particular need for increased quantities of this regulatory protein upon spore germination and resumption of active growth of plasmid-containing cells.
Ralstonia eutrophaH16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes
Authors: York Gregory M. , Lupberger Joachim , Tian Jiamin , Lawrence Adam G. , Stubbe JoAnne , Sinskey Anthony J. 
Published: June 2003

ABSTRACTIntracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) fromRalstonia eutrophawas identified. We now report identification of candidate PHB depolymerase genes fromR.eutropha, namely,phaZ2andphaZ3, and their characterization in vivo.phaZ1was used to identify two candidate depolymerase genes in the genome ofRalstonia metallidurans.phaZ1and these genes were then used to design degenerate primers. These primers and PCR methods on theR.eutrophagenome were used to identify two new candidate depolymerase genes inR.eutropha:phaZ2andphaZ3. Inverse PCR methods were used to obtain the complete sequence ofphaZ3, and library screening was used to obtain the complete sequence ofphaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generatingR.eutrophaH16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.
Identification and Mutational Analysis of Bacteriophage PRD1 Holin Protein P35
Authors: Rydman Pia S. , Bamford Dennis H. 
Published: June 2003

ABSTRACT

Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene
XXXV
). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene
S
), and the defect in PRD1 gene
XXXV
can be corrected by the presence of gene
S
of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene
XXXV
were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.
Molecular Analysis of the Copper-Transporting Efflux System CusCFBA of
Escherichia coli
Authors: Franke Sylvia , Grass Gregor , Rensing Christopher , Nies Dietrich H. 
Published: June 2003

ABSTRACT

The
cus
determinant of
Escherichia coli
encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux. CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance. Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance. Substitutions for several other methionine residues of this protein did not have any effect. The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein. CusF bound one copper per polypeptide. The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm. Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis. CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay. In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF. These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex.
Characterization of LytH, a Differentiation-Associated Peptidoglycan Hydrolase of
Bacillus subtilis
Involved in Endospore Cortex Maturation
Authors: Horsburgh Gavin J. , Atrih Abdelmadjid , Foster Simon J. 
Published: June 2003

ABSTRACT

The cortex peptidoglycan from endospores of
Bacillus subtilis
is responsible for the maintenance of dormancy. LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination. The
lytH
gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor σ
K
. Spores of a
lytH
mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy. Analysis of the peptidoglycan structure of
lytH
mutant spores shows the loss of muramic acid residues substituted with
l
-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain. In a
lytH cwlD
mutant, the lack of muramic acid residues substituted with
l
-alanine and δ-lactam leaves 97% of residues substituted with tetrapeptide. These results suggest that
lytH
encodes an
l
-Ala-
d
-Glu peptidase involved in production of single
l
-alanine side chains from tetrapeptides in the spore cortex. The lack of di- or tripeptides in a
lytH
mutant reveals the enzyme is an endopeptidase.
The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function
Authors: Enggist Elisabeth , Thöny-Meyer Linda 
Published: June 2003

ABSTRACT

CcmE is a heme chaperone active in the cytochrome
c
maturation pathway of
Escherichia coli
. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome
c
. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type
c
cytochromes, only one additional residue, D131, was required, and for efficient cytochrome
c
maturation, the seven-residue sequence
131
DENYTPP
137
was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome
c
maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.
Characterization of the
Vibrio
vulnificus
putAP
Operon, Encoding Proline Dehydrogenase and Proline Permease, and Its Differential Expression in Response to Osmotic Stress
Authors: Lee Jeong Hyun , Park Na Young , Lee Myung Hee , Choi Sang Ho 
Published: June 2003

ABSTRACT

The
Vibrio vulnificus putAP
genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction. Proline dehydrogenase activity and the level of
putA
transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down. Northern blotting and primer extension analyses revealed that transcription of
putAP
genes results in two different transcripts, transcript A (
putA
transcript) and transcript AP (
putAP
transcript). Expression of
putAP
genes was directed by two promoters, promoter P

putA

and promoter P

putAP

. A
crp
null mutation decreased proline dehydrogenase activity and the level of the
put
transcripts, indicating that transcription of
putAP
is under the positive control of cyclic AMP receptor protein. Proline dehydrogenase and the level of both
put
transcripts were increased by proline but repressed by glutamate. In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl. Since P

putA

activity, not P

putAP

activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through P

putA

and P

putAP

, respectively. Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in
V
.
vulnificus
.