bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an Effector Specificity Subregion within the Aromatic-Responsive Regulators DmpR and XylR by DNA Shuffling
Authors: Skärfstad Eleonore , O'Neill Eric , Garmendia Junkal , Shingler Victoria 
Published: July 2002

ABSTRACT

The
Pseudomonas
derived ς
54
-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.
Mre11 and Rad50 from
Pyrococcus furiosus
: Cloning and Biochemical Characterization Reveal an Evolutionarily Conserved Multiprotein Machine
Authors: Hopfner Karl-Peter , Karcher Annette , Shin David , Fairley Cecilia , Tainer John A. , Carney James P. 
Published: July 2002

ABSTRACT

The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon
Pyrococcus furiosus
and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the
P. furiosus
Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.
Covalent Linkage of Polyamines to Peptidoglycan in
Anaerovibrio lipolytica
Authors: Hirao Takashi , Sato Masahide , Shirahata Akira , Kamio Yoshiyuki 
Published: July 2002

ABSTRACT

Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of
Anaerovibrio lipolytica
, a strictly anaerobic bacterium. The peptidoglycan was degraded with the
N
-acetylmuramyl-
l
-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified as
l
-alanine–
d
-glutamic acid(
α
cadaverine)
γ
meso
-diaminopimelic acid (DAP)–
d
-alanine and
l
-alanine–
d
-glutamic acid(
α
spermidine)
γ
meso
-DAP–
d
-alanine, respectively. The N
1
-amino group of spermidine was linked to the α-carboxyl group of the
d
-glutamic acid residue of peptide II.
Genetic Evidence of Distinct Physiological Regulation Mechanisms in the ς
54
Pu
Promoter of
Pseudomonas putida
Authors: Cases Ildefonso , de Lorenzo Víctor 
Published: July 2002

ABSTRACT

The activity of the toluene-responsive ς
54
Pu
promoter of the pWW0 TOL plasmid of
Pseudomonas putida
is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. Although the
Pu
promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of β-galactosidase in a
Pu-lacZ
reporter
P. putida
strain. Since
Pu
was still down-regulated during exponential growth regardless of glucose addition, it appeared that the carbohydrate separately influenced promoter activity. This notion was supported by the growth-dependent induction pattern of
Pu
in a
ptsN
mutant of
P. putida
, the loss of which makes
Pu
no longer responsive to repression by glucose. On the other hand, overexpression of the sigma factor ς
54
, known to partially alleviate the exponential silencing of the promoter, did not affect glucose inhibition of
Pu
. These data indicated that exponential silencing and carbon source-dependent repression are two overlapping but genetically distinguishable mechanisms that adapt
Pu
to the physiological status of the cells and nutrient availability.
Methionine-to-Cysteine Recycling in
Klebsiella aerogenes
Authors: Seiflein Thomas A. , Lawrence Jeffrey G. 
Published: July 2002

ABSTRACT

In the enteric bacteria
Escherichia coli
and
Salmonella enterica
, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium
Klebsiella aerogenes
appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where
cys
mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in
E. coli
and
Salmonella
appear to be more intertwined in
Klebsiella
.
Identification of the Domains of UreR, an AraC-Like Transcriptional Regulator of the Urease Gene Cluster in
Proteus mirabilis
Authors: Poore Carrie A. , Coker Christopher , Dattelbaum Jonathan D. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

Proteus mirabilis
urease catalyzes the hydrolysis of urea to CO
2
and NH
3
, resulting in urinary stone formation in individuals with complicated urinary tract infections. UreR, a member of the AraC family, activates transcription of the genes encoding urease enzyme subunits and accessory proteins,
ureDABCEFG
, as well as its own transcription in the presence of urea. Based on sequence homology with AraC, we hypothesized that UreR contains both a dimerization domain and a DNA-binding domain. A translational fusion of the leucine zipper dimerization domain (amino acids 302 to 350) of C/EBP and the C-terminal half of UreR (amino acids 164 to 293) activated transcription from the
ureD
promoter (p

ureD

) and bound to a 60-bp fragment containing p

ureD

, as analyzed by gel shift. These results were consistent with the DNA-binding specificity residing in the C-terminal half of UreR and dimerization being required for activity. To localize the dimerization domain of UreR, a translational fusion of the DNA-binding domain of the LexA repressor (amino acids 1 to 87) and the N-terminal half of UreR (amino acids 1 to 182) was constructed and found to repress transcription from p

sulA

-
lac
Z (
sulA
is repressed by LexA) and bind to the
sulA
operator site, as analyzed by gel shift. Since LexA binds this site only as a dimer, the UreR
1–182
-LexA
1–87
fusion also must dimerize to bind p

sulA

. Indeed, purified UreR-
Myc
-His eluted from a gel filtration column as a dimer. Therefore, we conclude that the dimerization domain of UreR is located within the N-terminal half of UreR. UreR contains three leucines that mimic the leucines that contribute to dimerization of AraC. Mutagenesis of Leu147, Leu148, or L158 alone did not significantly affect UreR function. In contrast, mutagenesis of both Leu147 and Leu148 or all three Leu residues resulted in a 85 or 94% decrease, respectively, in UreR function in the presence of urea (
P
< 0.001). On the contrary, His102 and His175 mutations of UreR resulted in constitutive induction in the absence of urea. We conclude that a dimerization domain resides in the N-terminal half of the polypeptide, that Leu residues may contribute to this function, and that sequences within the C-terminal half of UreR are responsible for DNA binding to the urease promoter regions. Selected His residues also contribute significantly to UreR function.
Gonococcal MinD Affects Cell Division in
Neisseria gonorrhoeae
and
Escherichia coli
and Exhibits a Novel Self-Interaction
Authors: Szeto Jason , Ramirez-Arcos Sandra , Raymond Claude , Hicks Leslie D. , Kay Cyril M. , Dillon Jo-Anne R. 
Published: July 2002

ABSTRACT

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus
Neisseria gonorrhoeae
contains a
min
operon, and the present study investigates the role of
minD
from this operon. A gonococcal
minD
insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from
N. gonorrhoeae
(MinD
Ng
) in this mutant. Hence, MinD
Ng
is required for maintaining proper cell division and growth in
N. gonorrhoeae
. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD
Ng
is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC
Ng
and MinD
Ng
from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC
Ng
or MinD
Ng
alone did not display noticeable morphological changes. These studies suggest that MinD
Ng
is involved in inhibiting gonococcal cell division, likely in conjunction with MinC
Ng
. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD
Ng
in wild-type
Escherichia coli
led to cell filamentation, while overexpression in an
E. coli minD
mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD
Ng
revealed a novel MinD
Ng
self-interaction. We have also shown by yeast two-hybrid analysis that MinD from
E. coli
interacts with itself and with MinD
Ng
. These results indicate that MinD
Ng
is required for maintaining proper cell division and growth in
N. gonorrhoeae
and suggests that the self-interaction of MinD may be important for cell division site selection across species.
Transcriptional Analysis of the
tet
(P) Operon from
Clostridium perfringens
Authors: Johanesen Priscilla A. , Lyras Dena , Bannam Trudi L. , Rood Julian I. 
Published: July 2002

ABSTRACT

The
Clostridium perfringens
tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes,
tetA
(P) and
tetB
(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible
tetA
(P) and
tetB
(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the
tetA
(P) start codon. Deletion of P3 or alteration of the spacing between the −35 and −10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the
tetA
(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the
tet
(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.
Exchange of Xcp (Gsp) Secretion Machineries between
Pseudomonas aeruginosa
and
Pseudomonas alcaligenes
: Species Specificity Unrelated to Substrate Recognition
Authors: de Groot Arjan , Koster Margot , Gérard-Vincent Manon , Gerritse Gijs , Lazdunski Andrée , Tommassen Jan , Filloux Alain 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
and
Pseudomonas alcaligenes
are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12
xcp
gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with
Erwinia
, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175–190, 1996). In the present study, we report that XcpP and XcpQ of
P. alcaligenes
could not substitute for their respective
P. aeruginosa
counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when
P. alcaligenes xcpP
and
xcpQ
were expressed simultaneously in a
P. aeruginosa xcpPQ
deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable
P. alcaligenes
XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).
Myxococcus xanthus mokA
Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance
Authors: Kimura Yoshio , Nakano Hiromi , Terasaka Hideaki , Takegawa Kaoru 
Published: July 2002

ABSTRACT

A gene,
mokA
, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a
Myxococcus xanthus
genomic library. The predicted
mokA
gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain.
mokA
mutants placed under starvation conditions exhibited reduced sporulation. Mutation of
mokA
also caused marked growth retardation at high osmolarity. These results indicated that
M. xanthus
MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold
Dictyostelium discoideum.
Sequence Diversity and Molecular Evolution of the Leukotoxin (
lktA
) Gene in Bovine and Ovine Strains of
Mannheimia
(
Pasteurella
)
haemolytica
Authors: Davies Robert L. , Whittam Thomas S. , Selander Robert K. 
Published: July 2002

ABSTRACT

The molecular evolution of the leukotoxin structural gene (
lktA
) of
Mannheimia
(
Pasteurella
)
haemolytica
was investigated by nucleotide sequence comparison of
lktA
in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in
M. haemolytica
. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the
lktA
gene of the two related species
Mannheimia glucosida
and
Pasteurella trehalosi
, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess
lktA
alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of
lktA
in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.
Comparative Sequence Analysis of the Symbiosis Island ofMesorhizobium lotiStrain R7A
Authors: Sullivan John T. , Trzebiatowski Jodi R. , Cruickshank Ruth W. , Gouzy Jerome , Brown Steven D. , Elliot Rachel M. , Fleetwood Damien J. , McCallum Nadine G. , Rossbach Uwe , Stuart Gabriella S. , Weaver Julie E. , Webby Richard J. , de Bruijn Frans J. , Ronson Clive W. 
Published: July 2002

ABSTRACTTheMesorhizobium lotistrain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them toLotussymbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island ofM. lotistrain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include atrboperon and a cluster of potentialtragenes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to thevirpilus fromAgrobacterium tumefaciensthat is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.
An Adenylyl Cyclase, CyaA, of
Myxococcus xanthus
Functions in Signal Transduction during Osmotic Stress
Authors: Kimura Yoshio , Mishima Yukako , Nakano Hiromi , Takegawa Kaoru 
Published: July 2002

ABSTRACT

An adenylyl cyclase gene (
cyaA
) present upstream of an osmosensor protein gene (
mokA
) was isolated from
Myxococcus xanthus. cyaA
encoded a polypeptide of 843 amino acids with a predicted molecular mass of 91,187 Da. The predicted
cyaA
gene product had structural similarity to the receptor-type adenylyl cyclases that are composed of an amino-terminal sensor domain and a carboxy-terminal catalytic domain of adenylyl cyclase. In reverse transcriptase PCR experiments, the transcript of the
cyaA
gene was detected mainly during development and spore germination. A
cyaA
mutant, generated by gene disruption, showed normal growth, development, and germination. However, a
cyaA
mutant placed under conditions of ionic (NaCl) or nonionic (sucrose) osmostress exhibited a marked reduction in spore formation and spore germination. When wild-type and
cyaA
mutant cells at developmental stages were stimulated with 0.2 M NaCl or sucrose, the mutant cells increased cyclic AMP accumulation at levels similar to those of the wild-type cells. In contrast, the mutant cells during spore germination had mainly lost the ability to respond to high-ionic osmolarity. In vegetative cells, the
cyaA
mutant responded normally to osmotic stress. These results suggested that
M. xanthus
CyaA functions mainly as an ionic osmosensor during spore germination and that CyaA is also required for osmotic tolerance in fruiting formation and sporulation.
Inactivation of the Spirochete
recA
Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology
Authors: Kameni Ange-Patricia Tchamedeu , Couture-Tosi Evelyne , Saint-Girons Isabelle , Picardeau Mathieu 
Published: July 2002

ABSTRACT

Recently, we have shown the first evidence for allelic exchange in
Leptospira
spp. By using the same methodology, the cloned
recA
of
Leptospira biflexa
was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the
L. biflexa
chromosome by homologous recombination. The
recA
double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the
recA
mutant was about 10% of that of the parent strain. In addition, microscopic observation of the
L. biflexa recA
mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the
L. biflexa
wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the
recA
mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that
L. biflexa
RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.