bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTIntimins 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.