bluuebunny/crossref_metadata_2025_split huggingface dataset:
Safety and Shedding of an Attenuated Strain ofListeria monocytogeneswith a Deletion ofactA/plcBin Adult Volunteers: a Dose Escalation Study of Oral Inoculation
Authors: Angelakopoulos Haroula , Loock Katharina , Sisul David M. , Jensen Eric R. , Miller Jeff F. , Hohmann Elizabeth L. 
Published: July 2002

ABSTRACTListeria monocytogenesis an intracellular bacterial pathogen which causes bacteremia and has a tropism for the central nervous system and a propensity to cause maternofetal infection.L. monocytogeneshas been shown to be an effective prophylactic and a therapeutic vaccine vector for viral and tumor antigens in animal models.L. monocytogenesmutants lacking the ActA protein, which is essential for intracellular movement, are attenuated but retain immunogenicity in mice. Given the pathogenic potential ofL. monocytogenes, we created an attenuated mutant strain bearing double deletions in theactAandplcBvirulence genes for an initial clinical safety study of a prototypeL. monocytogenesvector in adults. Twenty healthy volunteers received single escalating oral doses (106to 109CFU, 4 volunteers per dose cohort) of this attenuatedL. monocytogenes, designated LH1169. Volunteers were monitored in the hospital for 14 days with frequent clinical checks and daily blood and stool cultures, and they were monitored for six additional weeks as outpatients. There were no positive blood cultures and no fevers attributable to the investigational inoculation. Most volunteers shed vaccine bacteria for 4 days or less, without diarrhea. One volunteer had a late positive stool culture during outpatient follow-up. Three volunteers had abnormal liver function test results temporally associated with inoculation; one could be reasonably attributed to another cause. In the highest-dose cohort, humoral, mucosal, and cellular immune responses to the investigational organism were detected in individual volunteers. AttenuatedL. monocytogenescan be studied in adult volunteers without serious long-term health sequelae.
Transcriptional Control of Expression of Genes for Photosynthetic Reaction Center and Light-Harvesting Proteins in the Purple Bacterium
Rhodovulum sulfidophilum
Authors: Masuda Shinji , Nagashima Kenji V. P. , Shimada Keizo , Matsuura Katsumi 
Published: July 2002

ABSTRACT

The purple photosynthetic bacterium
Rhodovulum sulfidophilum
synthesizes photosynthetic apparatus even under highly aerated conditions in the dark. To understand the oxygen-independent expression of photosynthetic genes, the expression of the
puf
operon coding for the light-harvesting 1 and reaction center proteins was analyzed. Northern blot hybridization analysis showed that
puf
mRNA synthesis was not significantly repressed by oxygen in this bacterium. High-resolution 5′ mapping of the
puf
mRNA transcriptional initiation sites and DNA sequence analysis of the
puf
upstream regulatory region indicated that there are three possible promoters for the
puf
operon expression, two of which have a high degree of sequence similarity with those of
Rhodobacter capsulatus
, which shows a high level of oxygen repression of photosystem synthesis. Deletion analysis showed that the third promoter is oxygen independent, but the activity of this promoter was not enough to explain the aerobic level of mRNA. The posttranscriptional
puf
mRNA degradation is not significantly influenced by oxygen in
R. sulfidophilum
. From these results, we conclude that
puf
operon expression in
R. sulfidophilum
is weakly repressed by oxygen, perhaps as a result of the following: (i) there are three promoters for
puf
operon transcription, at least one of which is oxygen independent; (ii) readthrough transcripts which may not be affected by oxygen may be significant in maintaining the
puf
mRNA levels; and (iii) the
puf
mRNA is fairly stable even under aerobic conditions.
Rapid Turnover of FlhD and FlhC, the Flagellar Regulon Transcriptional Activator Proteins, during
Proteus
Swarming
Authors: Claret Laurent , Hughes Colin 
Published: July 2002

ABSTRACT

The enterobacterial
flhDC
master operon activates expression of the flagellar biogenesis gene hierarchy and also represses cell division. During
Proteus mirabilis
differentiation into elongated hyperflagellated swarm cells,
flhDC
transcription is strongly but transiently increased. We show that concentration of the FlhD and FlhC proteins is also tightly controlled at the posttranslational level. This is achieved by protein degradation, which is most severe after differentiation when the half-life of both proteins is ca. 2 min. Degradation is energy dependent and putatively involves the Lon protease.
Cloning and Characterization of 1-Deoxy-
d
-Xylulose 5-Phosphate Synthase from
Streptomyces
sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis
Authors: Kuzuyama Tomohisa , Takagi Motoki , Takahashi Shunji , Seto Haruo 
Published: July 2002

ABSTRACT

In addition to the ubiquitous mevalonate pathway,
Streptomyces
sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-
d
-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene,
dxs
, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among
dxs
homologs from six genera. The
dxs
gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in
Escherichia coli
was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a
V
max
of 370 U per mg of protein and

K
m

s of 65 μM for pyruvate and 120 μM for
d
-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a

K
m

value of 35 mM for
d
-glyceraldehyde. To compare the enzymatic properties of CL190 and
E. coli
DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties.
The Right End of the
vir
Region of an Octopine-Type Ti Plasmid Contains Four New Members of the
vir
Regulon That Are Not Essential for Pathogenesis
Authors: Kalogeraki Virginia S. , Zhu Jun , Stryker Joel L. , Winans Stephen C. 
Published: July 2002

ABSTRACT

We sequenced the
virD-virE
,
virE-virF
, and
virF
–T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the
vir
gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensible for tumorigenesis.
Analysis of thebmpGene Family inBorrelia burgdorferiSensu Lato
Authors: Gorbacheva Victoria Y. , Godfrey Henry P. , Cabello Felipe C. 
Published: July 2002

ABSTRACTBmpA, BmpB, BmpC, and BmpD are homologousBorrelia burgdorferilipoproteins of unknown functions, encoded by thebmpgenes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of thebmpregion has been characterized for a variety ofB. burgdorferisensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All fourbmpgenes were present in the same relative order in allB. burgdorferisensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of thebmpgenes in these species, variations in DNA sequence in thebmpD-bmpCandbmpC-bmpAintergenic regions were significantly more common than in the corresponding 3′bmpDandbmpCcoding regions. The genetic structure of the chromosomal region containing thebmpgenes thus appears to be well conserved across different species ofB. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest thatbmpgene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced byB. burgdorferisensu lato.
Insertion Mutagenesis and Membrane Topology Model of the
Pseudomonas aeruginosa
Outer Membrane Protein OprM
Authors: Wong Kendy K. Y. , Hancock Robert E. W. 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite
Plasmodium falciparum
into 11 sites. In
Escherichia coli
, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a
P. aeruginosa
OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 β strands was proposed.
Quorum Sensing inVibrio fischeri: Analysis of the LuxR DNA Binding Region by Alanine-Scanning Mutagenesis
Authors: Egland Kristi A. , Greenberg E. P. 
Published: July 2002

ABSTRACTLuxR is the transcriptional activator for quorum-sensing control of luminescence inVibrio fischeri. A series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the DNA binding domain of LuxR was constructed, and the activity of each of the LuxR mutant proteins in recombinantEscherichia coliwas investigated. The region covered by the mutagenesis spanned residues 190 to 224. About half of the alanine-scanning mutants showed activities similar to that of the wild-type LuxR: at least two were positive-control mutants, four appeared to be defective in DNA binding, and several others were characterized as DNA binding affinity mutants. This analysis, taken together with information about other bacterial transcription factors, provides insights into amino acid residues in LuxR that are involved in DNA binding and transcriptional activation.
Role for
hetC
in the Transition to a Nondividing State during Heterocyst Differentiation in
Anabaena
sp
Authors: Xu Xudong , Wolk C. Peter 
Published: July 2002

ABSTRACT

Nitrogen-deprived filaments of wild-type or
hetC Anabaena
sp. produce respectively, at semiregular intervals, heterocysts and weakly fluorescent cells. Unlike heterocysts, the latter cells can divide and elongate, producing a pattern of spaced series of small cells. Because a
hetR
::
gfp
fusion is expressed most strongly in the small cells, we propose that these small cells represent a very early stage of heterocyst differentiation.
hetC
::
gfp
is expressed most strongly in proheterocysts and heterocysts.
Engineering a Homo-Ethanol Pathway in
Escherichia coli
: Increased Glycolytic Flux and Levels of Expression of Glycolytic Genes during Xylose Fermentation
Authors: Tao Han , Gonzalez Ramon , Martinez Alfredo , Rodriguez Maria , Ingram L. O. , Preston J. F. , Shanmugam K. T. 
Published: July 2002

ABSTRACT

Replacement of the native fermentation pathway in
Escherichia coli
B with a homo-ethanol pathway from
Zymomonas mobilis
(
pdc
and
adhB
genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (
P
< 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (
xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk,
and
pykA
) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes (
eno, fbaA, fbaB,
and
talA
) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes (
xylA
and
xylB
) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (
xylA
) and twofold higher for xylulokinase (
xylB
). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.
Forespore-Specific Transcription of the
lonB
Gene during Sporulation in
Bacillus subtilis
Authors: Serrano Monica , Hövel Sven , Moran Charles P. , Henriques Adriano O. , Völker Uwe 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
genome encodes two members of the Lon family of prokaryotic ATP-dependent proteases. One, LonA, is produced in response to temperature, osmotic, and oxidative stress and has also been implicated in preventing ς
G
activity under nonsporulation conditions. The second is encoded by the
lonB
gene, which resides immediately upstream from
lonA
. Here we report that transcription of
lonB
occurs during sporulation under ς
F
control and thus is restricted to the prespore compartment of sporulating cells. First, expression of a
lonB-lacZ
transcriptional fusion was abolished in strains unable to produce ς
F
but remained unaffected upon disruption of the genes encoding the early and late mother cell regulators ς
E
and ς
K
or the late forespore regulator ς
G
. Second, the fluorescence of strains harboring a
lonB-gfp
fusion was confined to the prespore compartment and depended on ς
F
production. Last, primer extension analysis of the
lonB
transcript revealed −10 and −35 sequences resembling the consensus sequence recognized by ς
F
-containing RNA polymerase. We further show that the
lonB
message accumulated as a single monocistronic transcript during sporulation, synthesis of which required ς
F
activity. Disruption of the
lonB
gene did not confer any discernible sporulation phenotype to otherwise wild-type cells, nor did expression of
lonB
from a multicopy plasmid. In contrast, expression of a fusion of the
lonB
promoter to the
lonA
gene severely reduced expression of the ς
G
-dependent
sspE
gene and the frequency of sporulation. In confirmation of earlier observations, we found elevated levels of ς
F
-dependent activity in a
spoIIIE47
mutant, in which the
lonB
region of the chromosome is not translocated into the prespore. Expression of either
lonB
or the P

lonB

-lonA
fusion from a plasmid in the
spoIIIE47
mutant reduced ς
F
-dependent activity to wild-type levels. The results suggest that both LonA and LonB can prevent abnormally high ς
F
activity but that only LonA can negatively regulate ς
G
.
Role of
ptsO
in Carbon-Mediated Inhibition of the
Pu
Promoter Belonging to the pWW0
Pseudomonas putida
Plasmid
Authors: Cases Ildefonso , Velázquez Francisco , de Lorenzo Vı́ctor 
Published: July 2002

ABSTRACT

An investigation was made into the role of the
ptsO
gene in carbon source inhibition of the
Pu
promoter belonging to the
Pseudomonas putida
upper TOL (toluene degradation) operon.
ptsO
is coexpressed with
ptsN
, the loss of which is known to render
Pu
unresponsive to glucose. Both
ptsN
and
ptsO
, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA
Ntr
and NPr, respectively, have been mapped adjacent to the
rpoN
gene of
P. putida
. The roles of these two genes in the responses of
Pu
to glucose were monitored by
lacZ
reporter technology with a
P. putida
strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking
ptsO
,
Pu
activity seemed to be inhibited even in the absence of glucose. A functional relationship with
ptsN
was revealed by the phenotype of a double
ptsN ptsO
mutant that was equivalent to the phenotype of a mutant with a single
ptsN
disruption. Moreover, phosphorylation of the product of
ptsO
seemed to be required for C inhibition of
Pu,
since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate
ptsO
revealed the presence of two open reading frames, designated
ptsP
and
mtp
, with the potential to encode PTS type I enzymes in
P. putida.
However, neither an insertion in
ptsP
nor an insertion in
mtp
resulted in a detectable change in inhibition of
Pu
by glucose. These results indicate that some PTS proteins have regulatory functions in
P. putida
that are independent of their recognized role in sugar transport in other bacteria.
Functional Analysis of OleY
l
-Oleandrosyl 3-
O
-Methyltransferase of the Oleandomycin Biosynthetic Pathway in
Streptomyces antibioticus
Authors: Rodrı́guez Leticia , Rodrı́guez David , Olano Carlos , Braña Alfredo F. , Méndez Carmen , Salas José A. 
Published: July 2002

ABSTRACT

Oleandomycin, a macrolide antibiotic produced by
Streptomyces antibioticus
, contains two sugars attached to the aglycon:
l
-oleandrose and
d
-desosamine.
oleY
codes for a methyltransferase involved in the biosynthesis of
l
-oleandrose. This gene was overexpressed in
Escherichia coli
to form inclusion bodies and in
Streptomyces lividans
, producing a soluble protein.
S. lividans
overexpressing
oleY
was used as a biotransformation host, and it converted the precursor
l
-olivosyl-erythronolide B into its 3-
O
-methylated derivative,
l
-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase:
l
-rhamnosyl- and
l
-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co
2+
. The
S. antibioticus
OleG2 glycosyltransferase is proposed to transfer
l
-olivose to the oleandolide aglycon, which is then converted into
l
-oleandrose by the OleY methyltransferase. This represents an alternative route for
l
-oleandrose biosynthesis from that in the avermectin producer
Streptomyces avermitilis
, in which
l
-oleandrose is transferred to the aglycon by a glycosyltransferase.
Disulfide Bond in
Pseudomonas aeruginosa
Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase
Authors: Liebeton Klaus , Zacharias Annette , Jaeger Karl-Erich 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from
Erwinia carotovora
were expressed in
Escherichia coli
, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of
P. aeruginosa
. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.
Escherichia coli
Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation
Authors: Pichoff Sebastien , Lutkenhaus Joe 
Published: July 2002

ABSTRACT

The
min
system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of
ftsZ
, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.