bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization of Two Chemotactic Transducers for Inorganic Phosphate in
Pseudomonas aeruginosa
Authors: Wu Hong , Kato Junichi , Kuroda Akio , Ikeda Tsukasa , Takiguchi Noboru , Ohtake Hisao
Published: July 2002

Two chemotactic transducers for inorganic phosphate (P
i
), designated CtpH and CtpL, have been identified in
Pseudomonas aeruginosa
. The corresponding genes (
ctpH
and
ctpL
) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the
P. aeruginosa
PAO1 genome. Computer-assisted capillary assays showed that the
ctpH
single mutant failed to exhibit P
i
taxis when the concentration of P
i
in the capillary was higher than 5 mM. Conversely, the
ctpL
single mutant could not respond to P
i
at the concentration of 0.01 mM. The
ctpH ctpL
double mutant was defective in P
i
taxis at any concentration ranging from 0.01 to 10 mM. To investigate regulation of P
i
taxis, the
ctpH
and
ctpL
genes were also disrupted individually in the
P. aeruginosa phoU
and
phoB
single mutants. The
ctpH phoU
and
ctpH phoB
double mutants were defective in P
i
taxis, regardless of whether the cells were starved for P
i
. The
ctpL phoU
double mutant was constitutive for P
i
taxis, whereas the
ctpL phoB
double mutant was induced by P
i
limitation for P
i
taxis. The region upstream of
ctpL
, but not
ctpH
, contained a putative
pho
box sequence. Expression of
ctpL
::
lacZ
was induced by P
i
limitation in PAO1, while it was constitutive in the
phoU
mutant. In contrast, the
phoB
mutant showed only background levels of
ctpL
::
lacZ
expression. These results showed that
ctpL
is involved in the
pho
regulon genes in
P. aeruginosa
. The
ctpH phoU
mutant, which failed to exhibit P
i
taxis, was constitutive for
ctpL
::
lacZ
expression, suggesting that the P
i
detection by CtpL requires PhoU. Like PAO1, the
phoB
and
phoU
single mutants were constitutive for expression of
ctpH
::
lacZ
. Thus, the evidence that the
ctpL phoU
mutant, but not the
ctpL phoB
mutant and PAO1, was constitutive for P
i
taxis raised the possibility that PhoU exerts a negative control on P
i
detection by CtpH at the posttranscriptional level.
Genetic Characterization of
Escherichia coli
Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype
Authors: Hamrick Terri S. , Harris Sandra L. , Spears Patricia A. , Havell Edward A. , Horton John R. , Russell Perry W. , Orndorff Paul E.
Published: July 2002

Five
Escherichia coli
type 1 pilus mutants that had point mutations in
fimH
, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts)
fimH
mutant that agglutinated guinea pig erythrocytes after growth at 31°C but not at 42°C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a
fimH
insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42°C but sensitive after growth at 31°C. The ts mutant thus binds macrophages with one receptor specificity at 31°C and another at 42°C.
SprE Levels Are Growth Phase Regulated in a ς
S
-Dependent Manner at the Level of Translation
Authors: Gibson Katherine E. , Silhavy Thomas J.
Published: July 2002

SprE regulates ς
S
levels in response to nutrient availability by promoting ClpXP-mediated degradation. Paradoxically, we observe that SprE is similarly regulated, accumulating preferentially upon starvation. This regulation of SprE levels is ς
S
dependent, altering SprE synthesis at the level of translation. Thus, we demonstrate that SprE and ς
S
function within a regulatory feedback loop.
Escherichia coli
DNA Polymerase IV Mutator Activity: Genetic Requirements and Mutational Specificity
Authors: Wagner Jérôme , Nohmi Takehiko
Published: July 2002

The
dinB
gene of
Escherichia coli
is known to be involved in the untargeted mutagenesis of λ phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F′ plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with
E. coli
UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase η) of
Saccharomyces cerevisiae
and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the
c
II gene of λ phage. The results indicated that the DNA Pol IV mutator activity is independent of
polA
,
polB
,
recA
,
umuDC
,
uvrA
, and
mutS
functions. The analysis of more than 300 independent mutations obtained in the wild-type or
mutS
background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5′-GX-3′ sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.
Dual Transcriptional Regulation of the
Escherichia coli
Phosphate-Starvation-Inducible
psiE
Gene of the Phosphate Regulon by PhoB and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Authors: Kim Soo-Ki , Kimura Sigenobu , Shinagawa Hideo , Nakata Atsuo , Lee Ki-Sung , Wanner Barry L. , Makino Kozo
Published: July 2002

We have shown that the
Escherichia coli
phosphate-starvation-inducible
psiE
gene is regulated by both phosphate and the carbon source by using both
lacZ
and chloramphenicol acetyltransferase gene (
cat
) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted −10 region was revealed by primer extension analysis. DNase I footprinting showed that the PhoB transcriptional-activator protein protects two predicted
pho
boxes lying upstream of and near the −35 promoter region. Similar analysis showed that the cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex binds a region that overlaps with the downstream
pho
box. These results, together with measurements of the in vivo
psiE
promoter activity under various conditions, show that expression of the
psiE
gene is under direct positive and negative control by PhoB and cAMP-CRP, respectively.
Transcriptional Regulation of the
cpr
Gene Cluster in
ortho
-Chlorophenol-Respiring
Desulfitobacterium dehalogenans
Authors: Smidt Hauke , van Leest Maarten , van der Oost John , de Vos Willem M.
Published: July 2002

To characterize the expression and possible regulation of reductive dehalogenation in halorespiring bacteria, a 11.5-kb genomic fragment containing the
o
-chlorophenol reductive dehalogenase-encoding
cprBA
genes of the gram-positive bacterium
Desulfitobacterium dehalogenans
was subjected to detailed molecular characterization. Sequence analysis revealed the presence of eight designated genes with the order
cprTKZEBACD
and with the same polarity except for
cprT
. The deduced
cprC
and
cprK
gene products belong to the NirI/NosR and CRP-FNR families of transcription regulatory proteins, respectively. CprD and CprE are predicted to be molecular chaperones of the GroEL type, whereas
cprT
may encode a homologue of the trigger factor folding catalysts. Northern blot analysis, reverse transcriptase PCR, and primer extension analysis were used to elucidate the transcriptional organization and regulation of the
cpr
gene cluster. Results indicated halorespiration-specific transcriptional induction of the monocistronic
cprT
gene and the biscistronic
cprBA
and
cprZE
genes. Occasional read-through at
cprC
gives rise to a tetracistronic
cprBACD
transcript. Transcription of
cprBA
was induced 15-fold upon addition of the
o
-chlorophenolic substrate 3-chloro-4-hydroxyphenylacetic acid within 30 min with concomitant induction of dehalogenation activity. Putative regulatory protein binding motifs that to some extent resemble the FNR box were identified in the
cprT-cprK
and
cprK-cprZ
intergenic regions and the promoter at
cprB
, suggesting a role for FNR-like CprK in the control of expression of the
cprTKZEBACD
genes.
radC102
of
Escherichia coli
Is an Allele of
recG
Authors: Lombardo Mary-Jane , Rosenberg Susan M.
Published: July 2002

The
radC102
mutation causes mild UV and X-ray sensitivity and was mapped previously to near
pyrE
and
recG
at 82 min on the
Escherichia coli
chromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615–625, 1984). We report that
radC102
has two striking phenotypes characteristic of
recG
mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with
ruv
mutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the
radC
and
recG
genes in
radC102
strains revealed that the
radC102
mutation creates a stop codon in
recG
that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the
radC
+
gene did not affect the UV sensitivity of a wild-type strain, a
radC102
strain, or a
recG258
::Tn
10
mini-
kan
strain. We conclude that
radC102
is an allele of
recG
and that the function of the RadC protein remains to be determined.
H-NS Controls
pap
and
daa
Fimbrial Transcription in
Escherichia coli
in Response to Multiple Environmental Cues
Authors: White-Ziegler Christine A. , Villapakkam Anuradha , Ronaszeki Karla , Young Sarah
Published: July 2002

A comparative study was completed to determine the influence of various environmental stimuli on the transcription of three different fimbrial operons in
Escherichia coli
and to determine the role of the histone-like protein H-NS in this environmental regulation. The fimbrial operons studied included the
pap
operon, which encodes pyelonephritis-associated pili (P pili), the
daa
operon, which encodes F1845 fimbriae, and the
fan
operon, which encodes K99 fimbriae. Using
lacZYA
transcriptional fusions within each of the fimbrial operons, we tested temperature, osmolarity, carbon source, rich medium, oxygen levels, pH, amino acids, solid medium, and iron concentration for their effects on fimbrial gene expression. Low temperature, high osmolarity, glucose as a carbon source, and rich medium repressed transcription of all three operons. High iron did not alter transcription of any of the operons tested, whereas the remaining stimuli had effects on individual operons. For the
pap
and
daa
operons, introduction of the
hns651
mutation relieved the repression, either fully or partially, due to low temperature, glucose as a carbon source, rich medium, and high osmolarity. Taken together, these data indicate that there are common environmental cues that regulate fimbrial transcription in
E. coli
and that H-NS is an important environmental regulator for fimbrial transcription in response to several stimuli.
Inducible Metabolism of Phenolic Acids inPediococcus pentosaceusIs Encoded by an Autoregulated Operon Which Involves a New Class of Negative Transcriptional Regulator
Authors: Barthelmebs Lise , Lecomte Bruno , Divies Charles , Cavin Jean-François
Published: July 2002

Pediococcus pentosaceusdisplays a substrate-inducible phenolic acid decarboxylase (PAD) activity onp-coumaric acid. Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of theLactobacillus plantarum pdcgene was used to screen aP. pentosaceusgenomic library in order to clone the corresponding gene of this bacteria. One clone detected with this probe displayed a low PAD activity. Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity withL. plantarumPDC enzyme. This ORF was identified as thepadAgene. A second ORF was located just downstream of thepadAgene and displayed 37% identity with the product of theBacillus subtilis yfiOgene. Subcloning, transcriptional analysis, and expression studies withEscherichia coliof these two genes under thepadAgene promoter, demonstrated that the genes are organized in an autoregulated bicistronic operonic structure and that the gene located upstream of thepadAgene encodes the transcriptional repressor of thepadAgene. Transcription of thispadoperon inP. pentosaceusis acid phenol dependent.
Role of the H Protein in Assembly of the Photochemical Reaction Center and Intracytoplasmic Membrane in
Rhodospirillum rubrum
Authors: Cheng Yongjian S. , Brantner Christine A. , Tsapin Alexandre , Collins Mary Lynne Perille
Published: July 2002

Rhodospirillum rubrum
is a model for the study of membrane formation. Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus. This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC). The proteins of the photochemical components are encoded by the
puf
operon (LHα, LHβ, RC-L, and RC-M) and by
puhA
(RC-H).
R. rubrum puf
interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent. The
puh
region was cloned, and DNA sequence determination identified open reading frames
bchL
and
bchM
and part of
bchH
;
bchHLM
encode enzymes of bacteriochlorophyll biosynthesis. A
puhA
/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation. Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly. This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane. In complemented strains, expression of
puh
occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector. The
puhA
gene alone was sufficient to restore phototrophic growth provided that recombination occurred.
Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage
Authors: Ma Yan , Ludden Paul W.
Published: July 2002

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.
Rhodospirillum rubrum
strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the
nifH
gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-
32
P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-
14
C]NAD individually upon UV irradiation, but most
14
C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-
14
C]NAD suggested that Arg 101 is not absolutely required for NAD binding.
Nitrogen or Sulfur Starvation Differentially Affects Phycobilisome Degradation and Expression of the
nblA
Gene in
Synechocystis
Strain PCC 6803
Authors: Richaud Catherine , Zabulon Gérald , Joder Annette , Thomas Jean-Claude
Published: July 2002

Nitrogen (N) limitation in cyanobacteria is well documented: a reduced growth rate is observed, accompanied by a cessation of phycobiliprotein synthesis and an ordered degradation of phycobilisomes (PBS). This leads to a dramatic bleaching phenomenon known as chlorosis. In
Synechococcus
strain PCC 7942, bleaching due to PBS degradation is also observed under sulfur (S) or phosphorus (P) limitation, and all three are under the control of the
nblA
gene product, a 59-amino-acid polypeptide which is overexpressed under N, S, and P starvation (J. L. Collier, and A. R. Grossman, EMBO J. 13:1039–1047, 1994). Cyanobase sequence data for
Synechocystis
strain PCC 6803 indicate the presence of two tandem open reading frames (sll0452 and sll0453) homologous to
nblA
. We cloned the two genes, identified a unique 5′ mRNA end suggestive of a single transcription start site, and studied
nblA
expression under conditions of N or S starvation by Northern hybridization: transcripts were detected only under N starvation (no signal is detected in replete medium or with S starvation), whether
nblA1
or
nblA2
was used as a probe. Mutations in
nblA1
and
nblA2
were constructed by insertion of a kanamycin cassette; both mutations were nonbleaching under N starvation.
Synechocystis
strain PCC 6803 does not bleach under S starvation, consistent with the absence of
nblA
induction in these conditions. These results were confirmed by analysis of the PBS components: sequential degradation of phycocyanin and associated linkers was observed only under conditions of N starvation. This indicates differences between
Synechocystis
strain PCC 6803 and
Synechococcus
strain PCC 7942 in their regulatory and signaling pathways leading to N- and S-starved phenotypes.
CAP1
, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of
Candida albicans
Authors: Bahn Yong-Sun , Sundstrom Paula
Published: July 2002

In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen
Candida albicans
exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (
HWP1
). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in
C. albicans
, we identified, cloned, characterized, and disrupted the
C. albicans CAP1
gene.
C. albicans
strains with inactivated
CAP1
budded in conditions that led to germ tube formation in isogenic strains with
CAP1
. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the
cap1/cap1
mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in
C. albicans
. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having
CAP1
were markedly diminished in the budding
cap1/cap1
mutant.
C. albicans
strains with deletions of both alleles of
CAP1
were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient
cap1/cap1
mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.
Dehalogenation, Denitration, Dehydroxylation, and Angular Attack on Substituted Biphenyls and Related Compounds by a Biphenyl Dioxygenase
Authors: Seeger Michael , Cámara Beatriz , Hofer Bernd
Published: July 2002

The attack by the
bph
-encoded biphenyl dioxygenase of
Burkholderia
sp. strain LB400 on a number of symmetrical
ortho
-substituted biphenyls or quasi
ortho
-substituted biphenyl analogues has been investigated. 2,2′-Difluoro-, 2,2′-dibromo-, 2,2′-dinitro-, and 2,2′-dihydroxybiphenyl were accepted as substrates. Dioxygenation of all of these compounds showed a strong preference for the semisubstituted pair of vicinal
ortho
and
meta
carbons, leading to the formation of 2′-substituted 2,3-dihydroxybiphenyls by subsequent elimination of HX (X = F, Br, NO
2
, or OH). All of these products were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenases of
Burkholderia
sp. strain LB400 or of
Rhodococcus globerulus
P6. Dibenzofuran and dibenzodioxin, which may be regarded as analogues of doubly
ortho
-substituted biphenyls or diphenylethers, respectively, were attacked at the “quasi
ortho
” carbon (the angular position 4a) and its neighbor. This shows that an aromatic ring-hydroxylating dioxygenase of class IIB is able to attack angular carbons. The catechols formed, 2,3,2′-trihydroxybiphenyl and 2,3,2′-trihydroxydiphenylether, were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenase. While angular attack by the biphenyl dioxygenase was the main route of dibenzodioxin oxidation, lateral dioxygenation leading to dihydrodiols was the major reaction with dibenzofuran. These results indicate that this enzyme is capable of hydroxylating
ortho
or angular carbons carrying a variety of substituents which exert electron-withdrawing inductive effects. They also support the view that the conversions of phenols into catechols by ring-hydroxylating dioxygenases, such as the transformation of 2,2′-dihydroxybiphenyl into 2,3,2′-trihydroxybiphenyl, are the results of di- rather than of monooxygenations. Lateral dioxygenation of dibenzofuran and subsequent dehydrogenation and extradiol dioxygenation by a number of biphenyl-degrading strains yielded intensely colored dead-end products. Thus, dibenzofuran can be a useful chromogenic indicator for the activity of the first three enzymes of biphenyl catabolic pathways.
Maximal Expression of Membrane-Bound Nitrate Reductase in
Paracoccus
Is Induced by Nitrate via a Third FNR-Like Regulator Named NarR
Authors: Wood Nicholas J. , Alizadeh Tooba , Bennett Scott , Pearce Joanne , Ferguson Stuart J. , Richardson David J. , Moir James W. B.
Published: July 2002

Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In
Paracoccus pantotrophus
, the enzyme system that catalyzes this reaction is encoded by the
narKGHJI
gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from
narK
is
narR
, a gene encoding a member of the FNR family of transcriptional activators.
narR
is transcribed divergently from the other
nar
genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and
narK
but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the ς
70
subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription.
narR
is also found in
Paracoccus denitrificans
, indicating that this species contains at least three FNR homologues.
Molecular Physiology of Sugar Catabolism in
Lactococcus lactis
IL1403
Authors: Even Sergine , Lindley Nic D. , Cocaign-Bousquet Muriel
Published: July 2002

The metabolic characteristics of
Lactococcus lactis
IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.
Novel Topology of BfpE, a Cytoplasmic Membrane Protein Required for Type IV Fimbrial Biogenesis in Enteropathogenic
Escherichia coli
Authors: Blank T. Eric , Donnenberg Michael S.
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) produces the bundle-forming pilus (BFP), a type IV fimbria that has been implicated in virulence, autoaggregation, and localized adherence to epithelial cells. The
bfpE
gene is one of a cluster of
bfp
genes previously shown to encode functions that direct BFP biosynthesis. Here, we show that an EPEC strain carrying a nonpolar mutation in
bfpE
fails to autoaggregate, adhere to HEp-2 cells, or form BFP, thereby demonstrating that BfpE is required for BFP biogenesis. BfpE is a cytoplasmic membrane protein of the GspF family. To determine the membrane topology of BfpE, we fused
bfpE
derivatives containing 3′ truncations and/or internal deletions to alkaline phosphatase and/or β-galactosidase reporter genes, whose products are active only when localized to the periplasm or cytoplasm, respectively. In addition, we constructed BfpE sandwich fusions using a dual alkaline phosphatase/β-galactosidase reporter cassette and analyzed BfpE deletion derivatives by sucrose density flotation gradient fractionation. The data from these analyses support a topology in which BfpE contains four hydrophobic transmembrane (TM) segments, a large cytoplasmic segment at its N terminus, and a large periplasmic segment near its C terminus. This topology is dramatically different from that of OutF, another member of the GspF family, which has three TM segments and is predominantly cytoplasmic. These findings provide a structural basis for predicting protein-protein interactions required for assembly of the BFP biogenesis machinery.
Structure-Function Analysis of BfpB, a Secretin-Like Protein Encoded by the Bundle-Forming-Pilus Operon of Enteropathogenic
Escherichia coli
Authors: Schmidt Sarah A. , Bieber David , Ramer Sandra W. , Hwang Jaiweon , Wu Cheng-Yen , Schoolnik Gary
Published: July 2002

Production of type IV bundle-forming pili by enteropathogenic
Escherichia coli
(EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of ≤65°C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.
Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium
Azoarcus evansii
Authors: Schühle Karola , Jahn Martina , Ghisla Sandro , Fuchs Georg
Published: July 2002

In the β-proteobacterium
Azoarcus evansii
, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a β-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by β-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.
O2R, a Novel Regulatory Element Mediating Rox1p-Independent O
2
and Unsaturated Fatty Acid Repression of
OLE1
in
Saccharomyces cerevisiae
Authors: Nakagawa Youji , Sugioka Shigemi , Kaneko Yoshinobu , Harashima Satoshi
Published: July 2002

Fatty acid desaturation catalyzed by fatty acid desaturases requires molecular oxygen (O
2
).
Saccharomyces cerevisiae
cells derepress expression of
OLE1
encoding Δ9 fatty acid desaturase under hypoxic conditions to allow more-efficient use of limited O
2
. It has been proposed that aerobic conditions lead to repression of
OLE1
by well-established O
2
-responsive repressor Rox1p, since putative binding sequences for Rox1p are present in the promoter of
OLE1
. However, we revealed in this study that disruption of
ROX1
unexpectedly did not affect the O
2
repression of
OLE1
, indicating that a Rox1p-independent novel mechanism operates for this repression. We identified by promoter deletion analysis the 50-bp O
2
-regulated (O2R) element in the
OLE1
promoter approximately 360 bp upstream of the start codon. Site-directed mutagenesis of the O2R element showed that the putative binding motif (5′-GATAA-3′) for the GATA family of transcriptional factors is important for O
2
repression. Anaerobic derepression of
OLE1
transcription was repressed by unsaturated fatty acids (UFAs), and interestingly the O2R element was responsible for this UFA repression despite not being included within the fatty acid-regulated (FAR) element previously reported. The fact that such a short 50-bp O2R element responds to both O
2
and UFA signals implies that O
2
and UFA signals merge in the ultimate step of the pathways. We discuss the differential roles of FAR and O2R elements in the transcriptional regulation of
OLE1.
Vertical Transmission of Biosynthetic Plasmids in Aphid Endosymbionts (
Buchnera
)
Authors: Wernegreen Jennifer J. , Moran Nancy A.
Published: July 2002

This study tested for horizontal transfer of plasmids among
Buchnera aphidicola
strains associated with ecologically and phylogenetically related aphid hosts (
Uroleucon
species). Phylogenetic congruence of
Buchnera
plasmid (
trpEG
and
leuABC
) and chromosomal (
dnaN
and
trpB
) genes supports strictly vertical long-term transmission of plasmids, which persist due to their contributions to host nutrition rather than capacity for infectious transfer. Synonymous divergences indicate elevated mutation on plasmids relative to chromosomal genes.
Functional Domains of Yeast Plasmid-Encoded Rep Proteins
Authors: Sengupta A. , Blomqvist K. , Pickett A. J. , Zhang Y. , Chew J. S. K. , Dobson M. J.
Published: July 2002

Both of theSaccharomyces cerevisiae2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector withrep1deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.
Loss of Ribosomal Protein L11 Blocks Stress Activation of the
Bacillus subtilis
Transcription Factor ς
B
Authors: Zhang Shuyu , Scott Janelle M. , Haldenwang W. G.
Published: July 2002

ς
B
, the general stress response sigma factor of
Bacillus subtilis
, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates ς
B
through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of ς
B
from an anti-ς
B
factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the ς
B
stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude
B. subtilis
extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of ς
B
.
B. subtilis
strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of ς
B
. Neither the energy-responsive activation of ς
B
nor stress-dependent chaperone gene induction (a ς
B
-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of ς
B
are shown to be active in the RplK
−
strain but fail to be triggered by stress. The data demonstrate that the
B. subtilis
ribosomes provide an essential input for the stress activation of ς
B
and suggest that the ribosomes may themselves be the sensors for stress in this system.
Low-Molecular-Weight Plasmid of
Salmonella enterica
Serovar Enteritidis Codes for Retron Reverse Transcriptase and Influences Phage Resistance
Authors: Rychlik I. , Sebkova A. , Gregorova D. , Karpiskova R.
Published: July 2002

Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that
Salmonella enterica
serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly,
Salmonella
serovar Enteritidis-produced low-molecular-weight DNA was shown to be a double-stranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19- and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis.
Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in
Ralstonia eutropha
JMP134
Authors: Louie Tai Man , Webster Christopher M. , Xun Luying
Published: July 2002

Ralstonia eutropha
JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH
2
)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (
tcpABC
) from JMP134 by using primers designed from conserved regions of FADH
2
-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that
tcpA
,
tcpB
, and
tcpC
encoded an FADH
2
-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The
tcpA
mutant failed to degrade 2,4,6-TCP, while both
tcpB
and
tcpC
mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of
tcpB
may have led to a polar effect on downstream
tcpC
, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH
2
was supplied by an
Escherichia coli
flavin reductase. TcpC produced in
E. coli
oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that
tcpB
may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.
Functional Analysis of HrpF, a Putative Type III Translocon Protein from
Xanthomonas campestris
pv. vesicatoria
Authors: Büttner Daniela , Nennstiel Dirk , Klüsener Birgit , Bonas Ulla
Published: July 2002

Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen
Xanthomonas campestris
pv. vesicatoria, encoded by the
hrp
(hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an
hrpF
null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.
Development of Monoclonal Antibodies Which Specifically RecognizeEntamoeba histolyticain Preserved Stool Samples
Authors: Yau Yvonne C. W. , Crandall Ian , Kain Kevin C.
Published: July 2002

We report the generation of monoclonal antibodies against a recombinant 170-kDa subunit of the Gal or GalNAc lectin ofEntamoeba histolyticathat specifically recognizeE. histolyticabut notEntamoeba disparin preserved stool samples. These antibodies do not cross-react with other bowel protozoa, includingEntamoeba coli,Giardia lamblia, andDientamoeba fragilis.
Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqMan Real-Time PCR Assay
Authors: Désiré Nathalie , Dehée Axelle , Schneider Véronique , Jacomet Christine , Goujon Christophe , Girard Pierre-Marie , Rozenbaum Willy , Nicolas Jean-Claude
Published: July 2002

Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log
10
. Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10
6
PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10
6
PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (
P
= 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5′ end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4
+
cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.
Combinatorial Use of Antibodies to Secreted Mycobacterial Proteins in a Host Immune System-Independent Test for Tuberculosis
Authors: Landowski Christopher P. , Godfrey Henry P. , Bentley-Hibbert Stuart I. , Liu Xinyan , Huang Zhishan , Sepulveda Ricardo , Huygen Kris , Gennaro Maria L. , Moy Fred H. , Lesley Scott A. , Haak-Frendscho Mary
Published: July 2002

Laboratory diagnosis of tuberculosis is often difficult. Immunodetection of circulating
Mycobacterium tuberculosis
proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays. We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S. I. Bentley-Hibbert, X. Quan, T. Newman, K. Huygen, and H. P. Godfrey, Infect. Immun. 67:581–588, 1999). To extend these observations to a
Mycobacterium bovis
BCG-vaccinated population and to another secreted mycobacterial protein, Ag85 and PstS-1 (protein antigen B, p38 antigen) were quantified in sera from 97 Chilean tuberculosis patients and healthy controls (many of whom had received BCG as children) using dot immunobinding, mouse monoclonal anti-BCG Ag85 complex antibody, and chicken antipeptide antibodies reactive with
M. tuberculosis
Ag85B and PstS-1. The latter antibodies had been raised to peptide-derived immunogens expressed on a novel proprietary protein carrier in
Escherichia coli
. Median serum Ag85 levels measured by using either anti-Ag85 antibody were significantly higher in patients with active tuberculosis than in healthy controls (
P
, <0.001 to 0.01); the median serum PstS-1 levels were similar in patients and controls. The sensitivity of significantly elevated circulating Ag85 levels in patients with pulmonary tuberculosis measured by anti-Ag85 complex or anti-Ag85B antibodies was 60 and 55%, respectively, but increased to 77% when results obtained with both anti-Ag85 antibodies were considered jointly (
P
< 0.02). The corresponding specificities for individual and joint consideration were 95, 85, and 80%, respectively. These results indicate that elevated Ag85 levels can be detected in patients with active tuberculosis even after BCG vaccination and suggest that combinatorial use of antibodies directed at different epitopes of this protein could provide a viable strategy for developing new host immune response-independent diagnostic tests for tuberculosis.
Serological Differentiation of Experimentally Induced
Candida dubliniensis
and
Candida albicans
Infections
Authors: Moragues Marı́a D. , Omaetxebarria Miren J. , Elguezabal Natalia , Bikandi Joseba , Quindós Guillermo , Coleman David C. , Pontón José
Published: July 2002

Using a rabbit model of systemic infection, we show that it is possible to differentiate infections caused by
Candida dubliniensis
and other
Candida
species by detecting the antibody response mounted by the infected animals. These results confirm our previous observation in a patient with
C. dubliniensis
candidemia and suggest that detection of
C. dubliniensis
-specific antibodies is useful in the diagnosis of invasive candidiasis caused by this yeast.