bluuebunny/crossref_metadata_2025 huggingface dataset:
Upper and Lower Respiratory Tract Infection byStreptococcus pneumoniaeIs Affected by Pneumolysin Deficiency and Differences in Capsule Type
Authors: Kadioglu Aras , Taylor Sally , Iannelli Francesco , Pozzi Gianni , Mitchell Tim J. , Andrew Peter W.
Published: July 2002

Pneumococci frequently colonize the upper respiratory tract, and these pneumococci are believed to act as a reservoir for infection of the lower respiratory tract and bacteremia. We investigated how the pneumococcal toxin pneumolysin affects the capacity of pneumococci to infect the upper and lower respiratory tract of the mouse. Wild-typeStreptococcus pneumoniaeserotype 2 and 3 strains, a serotype 2 pneumolysin-deficient mutant, and a serotype 2 mutant with the pneumolysin gene reinserted were used to study differences in colonization and disease. In addition, we also examined a pneumococcal chimeric mutant (capsule type switched from serotype 2 to serotype 3) to gain further insight into the role that capsule plays in nasopharyngeal infection. Absence of pneumolysin was found to be associated with significantly lower numbers of pneumococci in the nasopharynx, trachea, and lungs. Differences in pneumococcal capsule type were found to have significant effects on pneumococcal infection of the nasopharynx, trachea, and lungs. However, it was the combination of capsule type and genetic background that was important, and the influence of this combination varied with the site of infection. For example, in the nasopharynx the wild-type serotype 3 strain and the capsule-switched mutant behaved similarly, whereas in the lungs the mutant that was switched to serotype 3 survived less well than the wild-type serotype 3 strain. The combination of capsule type and genetic background also determined virulence. Thus, the wild-type serotype 3 strain was virulent, whereas the capsule-switched mutant was avirulent.
β-Defensin 1 Contributes to Pulmonary Innate Immunity in Mice
Authors: Moser Christian , Weiner Daniel J. , Lysenko Elena , Bals Robert , Weiser Jeffrey N. , Wilson James M.
Published: July 2002

Innate immunity serves as a first line defense in vertebrate organisms by providing an initial barrier to microorganisms and triggering antigen-specific responses. Antimicrobial peptides are thought to be effectors of innate immunity through their antibiotic activity and direct killing of microorganisms. Evidence to support this hypothesis in vertebrates is indirect, based on expression profiles and in vitro assays using purified peptides. Here we investigated the function of antimicrobial peptides in vivo using mice deficient in an antimicrobial peptide, mouse β-defensin-1 (mBD-1). We find that loss of mBD-1 results in delayed clearance ofHaemophilus influenzaefrom lung. These data demonstrate directly that antimicrobial peptides of vertebrates provide an initial block to bacteria at epithelial surfaces.
Phylogenetic Analysis and Prevalence of Urosepsis Strains of
Escherichia coli
Bearing Pathogenicity Island-Like Domains
Authors: Bingen-Bidois Martine , Clermont Olivier , Bonacorsi Stéphane , Terki Mustapha , Brahimi Naïma , Loukil Chawki , Barraud Dominique , Bingen Edouard
Published: July 2002

We characterized 100
Escherichia coli
urosepsis isolates from adult patients according to host compromise status by means of ribotyping, PCR phylogenetic grouping, and PCR detection of
papG
alleles and the virulence-related genes
sfa/foc, fyuA
,
irp
-
2
,
aer
,
hly, cnf
-
1
and
hra
. We also tested these strains for copies of
pap
and
hly
and their direct physical linkage with other virulence genes in an attempt to look for pathogenicity islands (PAIs) described for the archetypal uropathogenic strains J96, CFT073, and 536. Most of the isolates belonged to
E. coli
phylogenetic groups B2 and D and bore
papG
allele II,
aer
, and
fyuA
/
irp
-
2. papG
allele II-bearing strains were more common in noncompromised patients, while
papG
allele-negative strains were significantly more frequent in compromised patients. Fifteen ribotypes were identified. The three archetypal strains harbored different ribotypes, and only one-third of our urosepsis strains were genetically related to one of the archetypal strains. Three and 18 strains harbored three and two copies of
pap
, respectively, and 5 strains harbored two copies of
hly. papGIII
was physically linked to
hly, cnf
-
1
, and
hra
(reported to be PAI II
J96
-like genetic elements) in 14% of the strains. The PAI II
J96
-like domain was inserted within
pheR
tRNA in 11 strains and near
leuX
tRNA in 3 strains. Moreover, the colocalized genes
cnf
-
1
,
hra
, and
hly
were physically linked to
papGII
in four strains and to no
pap
gene in three strains.
papGII
and
hly
(reported to be PAI I
CFT073
-like genetic elements) were physically linked in 16 strains, pointing to a PAI I
CFT073
-like domain. Three strains contained both a PAI II
J96
-like domain and a PAI I
CFTO73
-like domain. Forty-two strains harbored
papGII
but not
hly
, in keeping with the presence of a PAI II
CFT073
-like domain. Only one strain harbored a PAI I
536
-like domain (
hly
only), and none harbored a PAI I
J96
-like domain (
papGI
plus
hly
) or a PAI II
536
-like domain (
papGIII
plus
hly
). This study provides new data on the prevalence and variability of physical genetic linkage between
pap
and certain virulence-associated genes that are consistent with their colocalization on archetypal PAIs.
Proteome Analysis of Secreted Proteins of the Gastric Pathogen
Helicobacter pylori
Authors: Bumann Dirk , Aksu Sevil , Wendland Meike , Janek Katharina , Zimny-Arndt Uschi , Sabarth Nicolas , Meyer Thomas F. , Jungblut Peter R.
Published: July 2002

Secreted proteins (the secretome) of the human pathogen
Helicobacter pylori
may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.
Candida albicans
Hyphal Formation and the Expression of the Efg1-Regulated Proteinases Sap4 to Sap6 Are Required for the Invasion of Parenchymal Organs
Authors: Felk Angelika , Kretschmar Marianne , Albrecht Antje , Schaller Martin , Beinhauer Sabine , Nichterlein Thomas , Sanglard Dominique , Korting Hans C. , Schäfer Wilhelm , Hube Bernhard
Published: July 2002

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen
Candida albicans
. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by
C. albicans
wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of
C. albicans
. Wild-type cells expressed a distinct pattern of
SAP
genes during i.p. infections. Within the first 72 h after infection,
SAP1
,
SAP2
,
SAP4
,
SAP5
,
SAP6
, and
SAP9
were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking
EFG1
had either noticeably reduced or higher expressed levels of
SAP4
to
SAP6
transcripts in vitro depending on the culture conditions. During infection,
efg1
mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of
SAP4
to
SAP6
transcripts. Mutants lacking
SAP1
to
SAP3
had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking
SAP4
to
SAP6
showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single
sap4
,
sap5
, and
sap6
and double
sap4
and -
6
,
sap5
and -
6
, and
sap4
and -
5
double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional
SAP6
showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by
SAP6
, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the
SAP
genes normally associated with the hyphal morphology.
A Common Step for Changing Cell Shape in Fruiting Body and Starvation-Independent Sporulation of
Myxococcus xanthus
Authors: Licking E. , Gorski L. , Kaiser D.
Published: July 2002

Myxococcus xanthus
can sporulate in either of two ways: at the end of the program of fruiting body development or after exposure of growing cells to certain reagents such as concentrated glycerol. Fruiting body sporulation requires starvation, while glycerol sporulation requires rapid growth, and since the two types of spores are structurally somewhat different, it has generally been assumed that the two processes are different. However, a Tn
5
Lac insertion mutation, Ω7536, has been isolated which simultaneously blocks the development of fruiting body spores as well as glycerol-induced spores. Both sporulation pathways are blocked in the mutant within the process that converts a rod-shaped cell into a spherical spore. The Ω7536 locus is expressed at the time of cell shape change appropriate to each process, early after glycerol induction and late after starvation induction. On the C-signal response pathway, it is possible to identify positions for the normal function of the Ω7536 locus and for the inducing stimulus from glycerol that are unique and consistent with the observations. Although the two sporulation pathways differ in certain respects, it is shown that they share at least one step for changing a rod-shaped cell into a spherical spore.
Elo1p-Dependent Carboxy-Terminal Elongation of C14:1Δ
9
to C16:1Δ
11
Fatty Acids in
Saccharomyces cerevisiae
Authors: Schneiter Roger , Tatzer Verena , Gogg Gabriela , Leitner Erich , Kohlwein Sepp Dieter
Published: July 2002

Saccharomyces cerevisiae
medium-chain acyl elongase (
ELO1
) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Δ
9
)-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Δ
11
fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Δ
11
was dependent on a functional
ELO1
gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (α-elongation). In wild-type cells, the C16:1Δ
11
elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (
ole1
Δ mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that
ole1
Δ mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Δ
11
) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Δ
9
) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.
The Membrane-Bound H
+
-ATPase Complex Is Essential for Growth of
Lactococcus lactis
Authors: Koebmann Brian J. , Nilsson Dan , Kuipers Oscar P. , Jensen Peter R.
Published: July 2002

The eight genes which encode the (F
1
F
o
) H
+
-ATPase in
Lactococcus lactis
subsp.
cremoris
MG1363 were cloned and sequenced. The genes were organized in an operon with the gene order
atpEBFHAGDC
; i.e., the order of
atpE
and
atpB
is reversed with respect to the more typical bacterial organization. The deduced amino acid sequences of the corresponding H
+
-ATPase subunits showed significant homology with the subunits from other organisms. Results of Northern blot analysis showed a transcript at approximately 7 kb, which corresponds to the size of the
atp
operon. The transcription initiation site was mapped by primer extension and coincided with a standard promoter sequence. In order to analyze the importance of the H
+
-ATPase for
L. lactis
physiology, a mutant strain was constructed in which the original
atp
promoter on the chromosome was replaced with an inducible nisin promoter. When grown on GM17 plates the resulting strain was completely dependent on the presence of nisin for growth. These data demonstrate that the H
+
-ATPase is essential for growth of
L. lactis
under these conditions.
Substrate Ambiguity of 3-Deoxy-
d
-
manno
-Octulosonate 8-Phosphate Synthase from
Neisseria gonorrhoeae
Revisited
Authors: Sheflyan Galina Ya , Sundaram Appavu K. , Taylor William P. , Woodard Ronald W.
Published: July 2002

Homogeneous, recombinant 3-deoxy-
d
-
manno
-octulosonate 8-phosphate synthase from
Neisseria gonorrhoeae
is shown to catalyze the formation of 3-deoxy-
d
-
manno
-octulosonate 8-phosphate from phosphoenolpyruvate and
d
-arabinose 5-phosphate as determined from
1
H-nuclear magnetic resonance analysis of the product. This enzyme does not catalyze the condensation of
d
-erythrose 4-phosphate and phosphoenolpyruvate to form 3-deoxy-
d
-
ribo
-heptulosonate 7-phosphate, as was previously reported (P. S. Subramaniam, G. Xie, T. Xia, and R. A. Jensen, J. Bacteriol. 180:119–127, 1998).
A Truncated Soluble
Bacillus
Signal Peptidase Produced in
Escherichia coli
Is Subject to Self-Cleavage at Its Active Site
Authors: van Roosmalen M. L. , Jongbloed J. D. H. , Kuipers A. , Venema G. , Bron S. , van Dijl J. M.
Published: July 2002

Soluble forms of
Bacillus
signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of
Escherichia coli.
Here, we show that the degradation of soluble forms of the
Bacillus
signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in
E. coli.
Second, the purified active soluble form of SipS displayed self-cleavage
in vitro
. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the −1, −3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.
Characterization of the Hydrogen-Deuterium Exchange Activities of the Energy-Transducing HupSL Hydrogenase and H
2
-Signaling HupUV Hydrogenase in
Rhodobacter capsulatus
Authors: Vignais Paulette M. , Dimon Bernard , Zorin Nikolay A. , Tomiyama Masamitsu , Colbeau Annette
Published: July 2002

Rhodobacter capsulatus
synthesizes two homologous protein complexes capable of activating molecular H
2
, a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H
2
sensor encoded by the
hupUV
genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the
hupSL
-encoded and the
hupUV
-encoded enzymes in the presence of D
2
and H
2
O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of
R. capsulatus
contains a gene homologous to
hoxH
, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal
hoxH
gene was inactivated and
hoxH
mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4.5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory
hypD
gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H
2
formed in exchange with D
2
by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D
2
, HD, and H
2
by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H
2
with benzyl viologen, produced H
2
with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H
2
signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.
The Large Resolvase TndX Is Required and Sufficient for Integration and Excision of Derivatives of the Novel Conjugative Transposon Tn
5397
Authors: Wang Hongmei , Mullany Peter
Published: July 2002

Tn
5397
is a novel conjugative transposon, originally isolated from
Clostridium difficile
. This element can transfer between
C. difficile
strains and to and from
Bacillus subtilis
. It encodes a conjugation system that is very similar to that of Tn
916
. However, insertion and excision of Tn
5397
appears to be dependent on the product of the element encoded gene
tndX
, a member of the large resolvase family of site-specific recombinases. To test the role of
tndX
, the gene was cloned and the protein was expressed in
Escherichia coli
. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn
5397
representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn
5397
targets from
C. difficile
and
B. subtilis
were still hot spots for the minitransposon insertion in
E. coli
.
Effects of Major Spore-Specific DNA Binding Proteins on
Bacillus subtilis
Sporulation and Spore Properties
Authors: Setlow Barbara , McGinnis Kelly A. , Ragkousi Katerina , Setlow Peter
Published: July 2002

Sporulation of a
Bacillus subtilis
strain (termed α
−
β
−
) lacking the majority of the α/β-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in α
−
β
−
strains. The sporulation defects in α
−
β
−
strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, α/β-type SASP but not by a mutant SASP that binds DNA poorly. Spores from α
−
β
−
strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in α
−
β
−
spores were abolished by the synthesis of normal levels of α/β-type SASP. These results indicate that α/β-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.
Vibrio cholerae
VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases
Authors: Butterton Joan R. , Choi Michael H. , Watnick Paula I. , Carroll Patricia A. , Calderwood Stephen B.
Published: July 2002

A 7.5-kbp fragment of chromosomal DNA downstream of the
Vibrio cholerae
vibriobactin outer membrane receptor,
viuA
, and the vibriobactin utilization gene,
viuB
, was recovered from a
Sau
3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the
Escherichia coli
enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor,
fepA
, and the utilization gene,
fes
, the cloned DNA was examined for the ability to restore siderophore synthesis to
E. coli ent
mutants. Cross-feeding studies demonstrated that an
E. coli entF
mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of
viuB
which encoded a deduced protein of greater than 2,158 amino acids, homologous to
Yersinia
sp. HMWP2,
Vibrio anguillarum
AngR, and
E. coli
EntF. A mutant with an in-frame deletion of this gene, named
vibF
, was created with classical
V. cholerae
strain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned
vibF
fragment restored vibriobactin synthesis to normal. The expression of the
vibF
promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the
V. cholerae fur
gene. Expression of
vibF
was not autoregulatory and neither affected nor was affected by the expression of
irgA
or
viuA
. The promoter of
vibF
was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the
E. coli
Fur binding consensus sequence. A footprint of purified
V. cholerae
Fur on the
vibF
promoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of
vibF
is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.
Disruption of the
Borrelia burgdorferi gac
Gene, Encoding the Naturally Synthesized GyrA C-Terminal Domain
Authors: Knight Scott W. , Kimmel Betsy J. , Eggers Christian H. , Samuels D. Scott
Published: July 2002

The C-terminal domain of the A subunit of DNA gyrase, which we term Gac, is naturally synthesized in
Borrelia burgdorferi
as an abundant DNA-binding protein. Full-length GyrA, which includes the C-terminal domain, is also synthesized by the spirochete and functions as a subunit of DNA gyrase. We have disrupted synthesis of Gac as an independent protein and demonstrated that it is not essential for growth in a coumarin-resistant background. We detected no alterations in DNA maintenance, condensation, or topology in
B. burgdorferi
lacking this small DNA-binding protein.
Mutation of
ndh
Genes Leads to Inhibition of CO
2
Uptake Rather than HCO
3
−
Uptake in
Synechocystis
sp. Strain PCC 6803
Authors: Ohkawa Hiroshi , Price G. Dean , Badger Murray R. , Ogawa Teruo
Published: July 2002

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into
ndhB
of wild-type (WT)
Synechocystis
sp. strain PCC 6803. In these mutants and a mutant (M55) lacking
ndhB
, CO
2
uptake was much more strongly inhibited than HCO
3
−
uptake, i.e., the activities of CO
2
and HCO
3
−
uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO
2
uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO
2
uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five
ndhD
genes in
Synechocystis
sp. strain PCC 6803. The mutant lacking
ndhD3
grew much more slowly than the WT at pH 6.5 under 50 ppm CO
2
, although other
ndhD
mutants grew like the WT under these conditions and showed low affinity for CO
2
uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.
Functional Analysis of theAgrobacterium tumefaciensT-DNA Transport Pore Protein VirB8
Authors: Kumar Renu B. , Das Anath
Published: July 2002

The VirB8 protein ofAgrobacterium tumefaciensis essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations invirB8by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement avirB8deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement thevirB8deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain,virB8S87Lpartially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8R107Pis defective in interactions with both VirB9 and VirB10. A second mutant VirB8S87Lis defective in interaction with VirB9.
Cloning and Characterization of the
Flavobacterium johnsoniae
Gliding Motility Genes
gldD
and
gldE
Authors: Hunnicutt David W. , McBride Mark J.
Published: July 2002

Cells of
Flavobacterium johnsoniae
move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of
F. johnsoniae
propel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for
F. johnsoniae
gliding motility,
gldA
,
gldB
, and
ftsX
, have recently been described. Tn
4351
mutagenesis was used to identify another gene,
gldD
, that is needed for gliding. Tn
4351
-induced
gldD
mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type
gldD
into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection.
gldD
codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function.
gldE
, which lies immediately upstream of
gldD
, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression of
gldE
partially suppressed the motility defects of a
gldB
point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to
Borrelia burgdorferi
TlyC and
Salmonella enterica
serovar Typhimurium CorC.
Multiple Genes for the Last Step of Proline Biosynthesis in
Bacillus subtilis
Authors: Belitsky Boris R. , Brill Jeanette , Bremer Erhard , Sonenshein Abraham L.
Published: July 2002

The complete
Bacillus subtilis
genome contains four genes (
proG, proH, proI,
and
comER
) with the potential to encode Δ
1
-pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (
proG, proH,
and
proI
) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.
Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from
Geobacter sulfurreducens
Authors: Kaufmann Franz , Lovley Derek R.
Published: July 2002

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in
Geobacter
species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of
Geobacter sulfurreducens
catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min
−1
· mg
−1
. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP
+
oxidoreductase activity and catalyzed the reduction of NADP
+
with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the
G. sulfurreducens
genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from
Moorella thermoacetica
, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.
Role of the
crc
Gene in Catabolic Repression of the
Pseudomonas putida
GPo1 Alkane Degradation Pathway
Authors: Yuste Luis , Rojo Fernando
Published: July 2002

Expression of the alkane degradation pathway encoded in the OCT plasmid of
Pseudomonas putida
GPo1 is induced in the presence of alkanes by the AlkS regulator, and it is down-regulated by catabolic repression. The catabolic repression effect reduces the expression of the two AlkS-activated promoters of the pathway, named
PalkB
and
PalkS2
. The
P. putida
Crc protein participates in catabolic repression of some metabolic pathways for sugars and nitrogenated compounds. Here, we show that Crc has an important role in the catabolic repression exerted on the
P. putida
GPo1 alkane degradation pathway when cells grow exponentially in a rich medium. Interestingly, Crc plays little or no role on the catabolic repression exerted by some organic acids in a defined medium, which shows that these two types of catabolic repression can be genetically distinguished. Disruption of the
crc
gene led to a six- to sevenfold increase in the levels of the mRNAs arising from the AlkS-activated
PalkB
and
PalkS2
promoters in cells growing exponentially in rich medium. This was not due to an increase in the half-lives of these mRNAs. Since AlkS activates the expression of its own gene and seems to be present in limiting amounts, the higher mRNA levels observed in the absence of Crc could arise from an increase in either transcription initiation or in the translation efficiency of the
alkS
mRNA. Both alternatives would lead to increased AlkS levels and hence to elevated expression of
PalkB
and
PalkS2
. High expression of
alkS
from a heterologous promoter eliminated catabolic repression. Our results indicate that catabolic repression in rich medium is directed to down-regulate the levels of the AlkS activator. Crc would thus modulate, directly or indirectly, the levels of AlkS.
Two Nonredundant SecA Homologues Function in Mycobacteria
Authors: Braunstein Miriam , Brown Amanda M. , Kurtz Sherry , Jacobs William R.
Published: July 2002

The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of
Mycobacterium tuberculosis
. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in
Escherichia coli
and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export—the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in
Mycobacterium smegmatis
, we demonstrate that
secA1
is an essential gene. In contrast,
secA2
can be deleted and is the first example of a nonessential
secA
homologue. The essential nature of
secA1
, which is consistent with the conserved Sec pathway, leads us to believe that
secA1
represents the equivalent of
E. coli secA
. The results of a phenotypic analysis of a Δ
secA2
mutant of
M. smegmatis
are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.
Display of Passenger Proteins on the Surface ofEscherichia coliK-12 by the EnterohemorrhagicE. coliIntimin EaeA
Authors: Wentzel Alexander , Christmann Andreas , Adams Thorsten , Kolmar Harald
Published: July 2002

Intimins are members of a family of bacterial adhesins from pathogenicEscherichia coliwhich specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagicE. coliO157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitorEcballium elateriumtrypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REIvwere displayed on the surface ofE. coliK-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at anambercodon residing within the truncatedeaeAgene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding ofE. coliK-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes ofE. coliand their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.
Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of
Pyrococcus furiosus
Ornithine Carbamoyltransferase
Authors: Roovers Martine , Sanchez Rony , Legrain Christianne , Glansdorff Nicolas
Published: July 2002

We have obtained mutants of
Pyrococcus furiosus
ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
Preferential Cleavage of Degradative Intermediates of
rpsT
mRNA by the
Escherichia coli
RNA Degradosome
Authors: Spickler Catherine , Stronge Victoria , Mackie George A.
Published: July 2002

RNase E, the principal RNase capable of initiating mRNA decay, preferentially attacks 5′-monophosphorylated over 5′-triphosphorylated substrates. Site-specific cleavage in vitro of the
rpsT
mRNA by RNase H directed by chimeric 2′-O-methyl oligonucleotides was employed to create truncated RNAs which are identical to authentic degradative intermediates. The rates of cleavage of two such intermediates by RNase E in the RNA degradosome are significantly faster (2.5- to 8-fold) than that of intact RNA. This verifies the preference of RNase E for degradative intermediates and can explain the frequent “all-or-none” behavior of mRNAs during the decay process.
Na
+
/Glutamine (Asparagine) Cotransport by
Staphylococcus lugdunensis
and
Corynebacterium amycolatum
Authors: Sarker Rafiquel I. , Maloney Peter C.
Published: July 2002

Staphylococcus lugdunensis
and
Corynebacterium amycolatum
each have a Na
+
/glutamine cotransporter that displays an ordered reaction sequence at the extracellular surface, with sodium binding (

K
m

of 6.5 mM) before glutamine (

K
m

of 50 μM). Asparagine is low-affinity substrate (

K
m

≈ 1 mM) for each system.
Purification and Characterization of PBP4a, a New Low-Molecular-Weight Penicillin-Binding Protein from
Bacillus subtilis
Authors: Duez Colette , Vanhove Marc , Gallet Xavier , Bouillenne Fabrice , Docquier Jean-Denis , Brans Alain , Frère Jean-Marie
Published: July 2002

Penicillin-binding protein 4a (PBP4a) from
Bacillus subtilis
was overproduced and purified to homogeneity. It clearly exhibits
dd
-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the
Actinomadura
sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (
k
2
/
K
) for the acylation of the essential serine by benzylpenicillin is 300,000 M
−1
s
−1
for the
Actinomadura
sp. strain R39 peptidase, 1,400 M
−1
s
−1
for
B. subtilis
PBP4a, and 7,000 M
−1
s
−1
for
Escherichia coli
PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (
k
2
/
K
= 46,000 M
−1
s
−1
). PBP4a is also much more thermostable than the R39 enzyme.
The
apeE
Gene of
Salmonella enterica
Serovar Typhimurium Is Induced by Phosphate Limitation and Regulated by
phoBR
Authors: Conlin Christopher A. , Tan Seng L. , Hu Huajun , Segar Todd
Published: July 2002

Mutations in
apeR
, a regulatory locus of the outer membrane esterase
apeE
from
Salmonella enterica
serovar Typhimurium, were shown to be alleles of the
pstSCAB-phoU
high-affinity phosphate transport operon. Expression of
apeE
was induced by phosphate limitation, and this induction required the
phoBR
phosphate regulatory system.
The
hetF
Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium
Nostoc punctiforme
Authors: Wong Francis C. Y. , Meeks John C.
Published: July 2002

A novel gene,
hetF
, was identified as essential for heterocyst development in the filamentous cyanobacterium
Nostoc punctiforme
strain ATCC 29133. In the absence of combined nitrogen,
hetF
mutants were unable to differentiate heterocysts, whereas extra copies of
hetF
in
trans
induced the formation of clusters of heterocysts. Sequences hybridizing to a
hetF
probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of
hetF
were similar to those of
hetR
, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription of
hetR
begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the
hetF
mutant, this increase in
hetR
transcription was delayed, and a
hetR
promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of
hetR
occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the
hetF
mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of
hetR
and localization of the HetR protein in differentiating heterocysts.
Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon,
Pyrobaculum calidifontis
VA1
Authors: Amo Taku , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2002

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon,
Pyrobaculum calidifontis
strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C. The enzyme exhibited a

K
m

value of 170 mM toward H
2
O
2
and a
k
cat
value of 2.9 × 10
4
s
−1
·subunit
−1
at 25°C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (

kat
Pc

). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium,
Thermus
sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of

kat
Pc

revealed that no orthologue genes were present on the archaeal genomes, including those from the “aerobic” (hyper)thermophilic archaea
Aeropyrum pernix
,
Sulfolobus solfataricus
, and
Sulfolobus tokodaii
. Therefore, Kat

Pc

can be considered a rare example of a manganese catalase from archaea.
Directed Evolution of Toluene
ortho
-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation
Authors: Canada Keith A. , Iwashita Sachiyo , Shim Hojae , Wood Thomas K.
Published: July 2002

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene
ortho
-monooxygenase (TOM) of
Burkholderia cepacia
G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in
Escherichia coli,
the variant TOM-Green degraded TCE (2.5 ± 0.3 versus 1.39 ± 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and
trans
-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ± 0.03 versus 0.029 ± 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ± 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the α-subunit of the hydroxylase, which appears to act as a smaller “gate” to the diiron active center. This hypothesis was supported by the ability of
E. coli
expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.