bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of
CIS
on Activity in
trans
of the Replication Initiator Protein of an IncB Plasmid
Authors: Praszkier J. , Murthy S. , Pittard A. J. 
Published: July 2002

ABSTRACT

RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in
cis
. The
cis
activity of RepA is thought to be mediated by
CIS
, a 166-bp region of DNA separating the coding region of
repA
from the origin of replication (
ori
) of pMU720. To investigate the
trans
activity of RepA, the
repA
gene, without its cognate
ori
, was cloned on a multicopy plasmid, pSU39. The
ori
on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-β-
d
-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned
ori
by RepA produced in
trans
from the pSU39 derivatives. The effect of
CIS
, when present either on the RepA-producing or the
ori
plasmid or both, on the efficiency of replication of the
ori
plasmid in vivo, was determined. The presence of
CIS
, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the
ori
plasmid. This inhibitory activity of
CIS
was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast,
CIS
had no effect when present on the
ori
plasmid. Initiation of replication from the
ori
in
trans
was independent of transcription into
CIS
.
Genetic Analysis of Sliding Motility in
Mycobacterium smegmatis
Authors: Recht Judith , Martínez Asunción , Torello Sandra , Kolter Roberto 
Published: July 2002

ABSTRACT

A screen for nonsliding mutants of
Mycobacterium smegmatis
yielded 20 mutants with transposon insertions in the
mps
gene, which is involved in glycopeptidolipid biosynthesis. One mutant had an insertion in a gene predicted to encode a membrane transport protein. All mutants lacked glycopeptidolipids and were unable to form biofilms on polyvinyl chloride.
Characterization of
Escherichia coli
DNA Lesions Generated within J774 Macrophages
Authors: Schlosser-Silverman Eliana , Elgrably-Weiss Maya , Rosenshine Ilan , Kohen Ron , Altuvia Shoshy 
Published: July 2002

ABSTRACT

Macrophages are armed with multiple oxygen-dependent and -independent bactericidal properties. However, the respiratory burst, generating reactive oxygen species, is believed to be a major cause of bacterial killing. We exploited the susceptibility of
Escherichia coli
in macrophages to characterize the effects of the respiratory burst on intracellular bacteria. We show that
E. coli
strains recovered from J774 macrophages exhibit high rates of mutations. We report that the DNA damage generated inside macrophages includes DNA strand breaks and the modification 8-oxo-2′-deoxyguanosine, which are typical oxidative lesions. Interestingly, we found that under these conditions, early in the infection the majority of
E. coli
cells are viable but gene expression is inhibited. Our findings demonstrate that macrophages can cause severe DNA damage to intracellular bacteria. Our results also suggest that protection against the macrophage-induced DNA damage is an important component of the bacterial defense mechanism within macrophages.
Varying the Abundance of O Antigen in
Rhizobium etli
and Its Effect on Symbiosis with
Phaseolus vulgaris
Authors: Noel K. Dale , Forsberg Lennart S. , Carlson Russell W. 
Published: July 2002

ABSTRACT

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of
Rhizobium etli
mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn
5
insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the
R. etli
CE3
lps
genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host
Phaseolus vulgaris
, whereas the mutant suppressed by DNA from
lps
region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other
R. etli
mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on
P. vulgaris
. The latter strains were mutated in
lps
region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.
Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation
Authors: Hugo N. , Meyer C. , Armengaud J. , Gaillard J. , Timmis K. N. , Jouanneau Y. 
Published: July 2002

ABSTRACT

The
xylT
gene product, a component of the xylene catabolic pathway of
Pseudomonas putida
mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in
Escherichia coli
, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were −286 mV and −323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an
M
r
of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing
xylT
or its analogues were introduced into an XylT-null mutant of
P. putida
which is unable to grow on
p
-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.
The
nrfA
and
nirB
Nitrite Reductase Operons in
Escherichia coli
Are Expressed Differently in Response to Nitrate than to Nitrite
Authors: Wang Henian , Gunsalus Robert P. 
Published: July 2002

ABSTRACT

Escherichia coli
possesses two distinct nitrite reductase enzymes encoded by the
nrfA
and
nirB
operons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using
nrfA-lacZ
and
nirB-lacZ
reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimal
nrfA-lacZ
expression occurred only at low to intermediate levels of nitrate and where
nirB-lacZ
expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed
nrfA
gene expression. While either NarL or NarP was able to induce
nrfA-lacZ
expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by
nirBDC
under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress
nirB
expression. Nitrite was also an inducer of
nirB
and
nrfA
gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since
nrfA
operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the
nirB
nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.
Polyamine Transport and Role of
potE
in Response to Osmotic Stress in
Escherichia coli
Authors: Schiller Dirk , Kruse Daniela , Kneifel Helmut , Krämer Reinhard , Burkovski Andreas 
Published: July 2002

ABSTRACT

When transport of polyamines in
Escherichia coli
was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock. Spermidine was not excreted. Characterization of a deletion mutant showed that PotE is not involved in these transport processes.
Characterization of SotA and SotB, Two
Erwinia chrysanthemi
Proteins Which Modify Isopropyl-β-
d
-Thiogalactopyranoside and Lactose Induction of the
Escherichia coli lac
Promoter
Authors: Condemine Guy 
Published: July 2002

ABSTRACT

The expression, in
Escherichia coli
, of variants of the
Erwinia chrysanthemi
secretion genes
outB
and
outS
under the P
tac
promoter is toxic to the cells. During attempts to clone
E. chrysanthemi
genes able to suppress this toxicity, I identified two genes,
sotA
and
sotB
, whose products are able to reduce the isopropyl-β-
d
-thiogalactopyranoside (IPTG) induction of the
E. coli lac
promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of
sotA
and
sotB
is not regulated by their substrates, but
sotA
is activated by the cyclic AMP receptor protein (CRP), while
sotB
is repressed by CRP. Lactose is weakly toxic for
E. chrysanthemi
. This toxicity is increased in a
sotB
mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.
The TetA(K) Tetracycline/H
+
Antiporter from
Staphylococcus aureus
: Mutagenesis and Functional Analysis of Motif C
Authors: Ginn Samantha L. , Brown Melissa H. , Skurray Ronald A. 
Published: July 2002

ABSTRACT

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from
Staphylococcus aureus
; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H
+
activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.
Purification and Characterization of the DeoR Repressor of
Bacillus subtilis
Authors: Zeng Xianmin , Saxild Hans H. , Switzer Robert L. 
Published: July 2002

ABSTRACT

Transcription of the
Bacillus subtilis dra-nupC-pdp
operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in
Escherichia coli
and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the
dra-nupC-pdp
operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.
Characterization of the Gene Cluster Involved in Isoprene Metabolism in
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Leemhuis Hans , Spelberg Jeffrey H. Lutje , Janssen Dick B. 
Published: July 2002

ABSTRACT

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in
Rhodococcus
sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione
S
-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (
isoI
) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (
isoH
). Furthermore, a gene encoding a second glutathione
S
-transferase was identified (
isoJ
). The
isoJ
gene was overexpressed in
Escherichia coli
and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1,2-epoxy-2-methyl-3-butene. Downstream of
isoJ
, six genes (
isoABCDEF
) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from
Xanthobacter
sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (
isoG
) showed significant similarity with that of α-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.
areCBA
Is an Operon in
Acinetobacter
sp. Strain ADP1 and Is Controlled by AreR, a ς
54
-Dependent Regulator
Authors: Jones Rheinallt M. , Williams Peter A. 
Published: July 2002

ABSTRACT

The
areCBA
genes in
Acinetobacter
sp. strain ADP1, determining growth on benzyl alkanoates, are shown to be transcribed as a single operon and regulated by
areR
, which encodes a regulatory protein of the NtrC/XylR family. Assays of the Are enzymes and of two insertions of
lacZ
as a reporter gene have shown that the operon is induced by benzyl acetate, benzyl alcohol, and benzaldehyde, as well as 2- and 4-hydroxybenzyl acetates and benzyl propionate and butyrate. Two adjacent sites of transcriptional initiation were 97 and 96 bp upstream of the start codon for
areC
, near a ς
54
-dependent −12, −24 promoter. Inactivation of
areR
and
rpoN
(for RNA polymerase ς
54
) drastically reduced growth rates on the Are substrates and induction of the operon.
Reverse Genetics of
Escherichia coli
Glycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo
Authors: Holtman C. Kay , Pawlyk Aaron C. , Meadow Norman D. , Pettigrew Donald W. 
Published: July 2002

ABSTRACT

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of
Escherichia coli
glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA
Glc
(formerly known as III
glc
), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the
glpK
gene. Genetics methods based on homologous recombination are used to place
glpK
alleles with known specific mutations into the chromosomal context of the
glpK
gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The
E. coli
strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different
glpR
alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the
glpK
gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIA
Glc
inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.
HreP, an In Vivo
-
Expressed Protease of
Yersinia enterocolitica
, Is a New Member of the Family of Subtilisin/Kexin-Like Proteases
Authors: Heusipp Gerhard , Young Glenn M. , Miller Virginia L. 
Published: July 2002

ABSTRACT

The role of proteases in pathogenesis is well established for several microorganisms but has not been described for
Yersinia enterocolitica
. Previously, we identified a gene,
hreP
, which showed significant similarity to proteases in a screen for chromosomal genes of
Y. enterocolitica
that were exclusively expressed during an infection of mice. We cloned this gene by chromosome capture and subsequently determined its nucleotide sequence. Like
inv
, the gene encoding the invasin protein of
Y. enterocolitica
,
hreP
is located in a cluster of flagellum biosynthesis and chemotaxis genes. The genomic organization of this chromosomal region is different in
Escherichia coli, Salmonella,
and
Yersinia pestis
than in
Y. enterocolitica.
Analysis of the distribution of
hreP
between different
Yersinia
isolates and the relatively low G+C content of the gene suggests acquisition by horizontal gene transfer. Sequence analysis also revealed that HreP belongs to a family of eukaryotic subtilisin/kexin-like proteases. Together with the calcium-dependent protease PrcA of
Anabaena variabilis
, HreP forms a new subfamily of bacterial subtilisin/kexin-like proteases which might have originated from a common eukaryotic ancestor. Like other proteases of this family, HreP is expressed with an N-terminal prosequence. Expression of an HreP-His
6
tag fusion protein in
E. coli
revealed that HreP undergoes autocatalytic processing at a consensus cleavage site of subtilisin/kexin-like proteases, thereby releasing the proprotein.
Analysis of Promoter Recognition In Vivo Directed by ς
F
of
Bacillus subtilis
by Using Random-Sequence Oligonucleotides
Authors: Amaya Edward , Khvorova Anastasia , Piggot Patrick J. 
Published: July 2002

ABSTRACT

Formation of spores from vegetative bacteria by
Bacillus subtilis
is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase ς factors. Of these, ς
F
is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing ς
F
(E-ς
F
), and only two genes of known function are exclusively under the control of E-ς
F
,
spoIIR
and
spoIIQ
. In order to investigate the features of promoters that are recognized by E-ς
F
, we studied the effects of randomizing sequences for the −10 and −35 regions of the promoter for
spoIIQ
. The randomized promoter regions were cloned in front of a promoterless copy of
lacZ
in a vector designed for insertion by double crossover of single copies of the promoter-
lacZ
fusions into the
amyE
region of the
B. subtilis
chromosome. This system made it possible to test for transcription of
lacZ
by E-ς
F
in vivo. The results indicate a weak ς
F
-specific −10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated ς factors, as well as to ς
A
. There was a rather stronger −35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated ς factors. The looseness of the ς
F
promoter requirement contrasts with the strict requirement for ς
A
-directed promoters of
B. subtilis
. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-ς
F
in vivo.
EfficientvirGene Induction inAgrobacterium tumefaciensRequiresvirA,virG,andvirBox from the Same Ti Plasmid
Authors: Krishnamohan Atmakuri , Balaji V. , Veluthambi K. 
Published: July 2002

ABSTRACTThevirgenes of octopine, nopaline, andl,l-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopalinevircomponents. The induction of an octopinevirEA6::lacZfusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopinevirGA6in a nopaline strain with pSM358 did not completely restorevirEA6induction. However, addition of the octopinevirAA6to the above strain increasedvirEA6induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopalinevirEC58::catfusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) andl,l-succinamopine (A281) strains. Supplementation of nopalinevirAC58andvirGC58in an octopine strain (A348) harboring pUCD1553 increased induction levels ofvirEC58::catfusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine andl,l-succinamopine VirG proteins induce the octopinevirEA6more efficiently than they do the nopalinevirEC58. Conversely, the nopaline VirG protein induces the nopalinevirEC58more efficiently than it does the octopinevirEA6. The ability of Bo542virGto bring about supervirulence in tobacco is observed for an octopinevirhelper (LBA4404) but not for a nopalinevirhelper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG andvirboxes of different Ti plasmids. Efficientvirgene induction in octopine and nopaline strains requiresvirA,virG,andvirboxes from the respective Ti plasmids.