bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conservation, Surface Exposure, and In Vivo Expression of the Frp Family of Iron-Regulated Cell Wall Proteins in
Staphylococcus aureus
Authors: Morrissey Julie A. , Cockayne Alan , Hammacott Jane , Bishop Keith , Denman-Johnson Amy , Hill Philip J. , Williams Paul 
Published: July 2002

ABSTRACT

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic
Staphylococcus aureus
cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated
frpA
and
frpB
, respectively. Studies with an
S. aureus fur
mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative
S. aureus
membrane protein, FrpC.
frpC
is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of
S. aureus
under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of
S. aureus
to plastic in vitro. Binding of
S. aureus
to microtiter wells was found to be iron regulated, and iron-restricted
S. aureus
containing antisense
frpA
or
frpAB
but not
frpB
constructs showed reduced binding compared to vector construct controls.
Disruption of the Salmonella-Containing Vacuole Leads to Increased Replication of
Salmonella enterica
Serovar Typhimurium in the Cytosol of Epithelial Cells
Authors: Brumell John H. , Tang Patrick , Zaharik Michelle L. , Finlay B. Brett 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the
Salmonella
-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this
Salmonella
pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of
sifA
mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.
DNA Microarray Assessment of Putative
Borrelia burgdorferi
Lipoprotein Genes
Authors: Liang Fang Ting , Nelson F. Kenneth , Fikrig Erol 
Published: July 2002

ABSTRACT

A DNA microarray containing fragments of 137
Borrelia burgdorferi
B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from
B. burgdorferi
sensu stricto B31, 297, and N40;
Borrelia garinii
IP90; and
Borrelia afzelii
P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes encoding
B. burgdorferi
lipoproteins.
Cysteine Protease Activity and Histamine Release from the Human Mast Cell Line HMC-1 Stimulated by Recombinant Streptococcal Pyrogenic Exotoxin B/Streptococcal Cysteine Protease
Authors: Watanabe Yukino , Todome Yuko , Ohkuni Hisashi , Sakurada Shinsaku , Ishikawa Toshio , Yutsudo Takashi , Fischetti Vincent A. , Zabriskie John B. 
Published: July 2002

ABSTRACT

We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (
spe b
) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.
Real-Time Imaging of Fluorescent Flagellar Filaments
Authors: Turner Linda , Ryu William S. , Berg Howard C. 
Published: July 2002

ABSTRACT

Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of
Escherichia coli
. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.
Cloning and Expression of
ntnD
, Encoding a Novel NAD(P)
+
-Independent 4-Nitrobenzyl Alcohol Dehydrogenase from
Pseudomonas
sp. Strain TW3
Authors: James Keith D. , Hughes Michelle A. , Williams Peter A. 
Published: July 2002

ABSTRACT

Pseudomonas
sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (
ntnD
) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported
ntn
gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)
+
independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)
+
-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that
ntnD
is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other
ntn
genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that
ntnD
is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the
ntn
genes suggests that one or more recombination events have been involved in the formation of their current organization.
VirB6 Is Required for Stabilization of VirB5 and VirB3 and Formation of VirB7 Homodimers in
Agrobacterium tumefaciens
Authors: Hapfelmeier Siegfried , Domke Natalie , Zambryski Patricia C. , Baron Christian 
Published: July 2002

ABSTRACT

VirB6 from
Agrobacterium tumefaciens
is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of
virB6
in
trans
restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in
trans
. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a
virB6
deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.
A Carboxy-Terminal 16-Amino-Acid Region of ς
38
of
Escherichia coli
Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo
Authors: Ohnuma Mio , Fujita Nobuyuki , Ishihama Akira , Tanaka Kan , Takahashi Hideo 
Published: July 2002

ABSTRACT

ς
38
(or ς
S
, the
rpoS
gene product) is a sigma subunit of RNA polymerase in
Escherichia coli
and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς
38
(residues 315 to 330), which is well conserved among the
rpoS
gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-
lacZ
fusion constructs, but the mutant ς
38
retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς
38
holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.
Molecular Characterization of the β-N-Acetylglucosaminidase ofEscherichia coliand Its Role in Cell Wall Recycling
Authors: Cheng Qiaomei , Li Hongshan , Merdek Keith , Park James T. 
Published: July 2002

ABSTRACTThe β-N-acetylglucosaminidase ofEscherichia coliwas found to have a novel specificity and to be encoded by a gene (nagZ) that maps at 25.1 min. It corresponds to an open reading frame,ycfO, whose predicted amino acid sequence is 57% identical to that ofVibrio furnissiiExoII. NagZ hydrolyzes the β-1,4 glycosidic bond betweenN-acetylglucosamine andanhydro-N-acetylmuramic acid in cell wall degradation products following their importation into the cell during the process for recycling cell wall muropeptides. From amino acid sequence comparisons, the novel β-N-acetylglucosaminidase appears to be conserved in all 12 gram-negative bacteria whose complete or partial genome sequence data are available.
The Global Carbon Metabolism Regulator Crc Is a Component of a Signal Transduction Pathway Required for Biofilm Development by
Pseudomonas aeruginosa
Authors: O'Toole George A. , Gibbs Karine A. , Hager Paul W. , Phibbs Paul V. , Kolter Roberto 
Published: July 2002

ABSTRACT

The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in
Pseudomonas aeruginosa
. Using phase-contrast microscopy, we found that a
crc
mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation,
crc
mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in
pilA
(pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.
Organization and Expression of a
Thermus thermophilus
Arginine Cluster: Presence of Unidentified Open Reading Frames and Absence of a Shine-Dalgarno Sequence
Authors: Sanchez Rony , Roovers Martine , Glansdorff Nicolas 
Published: July 2002

ABSTRACT

A group of genes regulated by arginine was found clustered in the order
argF
-ORF1-
argC
-
argJ
-ORF4 between other, as yet uncharacterized, open reading frames (ORFs). Transcription starts were identified immediately upstream from
argF
and ORF4. Arginine repressed transcription that was initiated at
argF
but induced transcription of ORF4. The functions of ORF1 and ORF4 are unknown, but analysis of the sequence of ORF4 suggests that it is a membrane protein, possibly involved in transport of arginine or a related metabolite. Mobility shift and DNase I footprinting have revealed specific binding of pure
Escherichia coli
ArgR to the promoter region of
Thermus thermophilus argF
. These results suggest that
argF
transcription is controlled by a repressor homologous to those characterized in enteric bacteria and bacilli.
Thermus argF
mRNA is devoid of Shine-Dalgarno (SD) sequences. However, downstream from the ATG start codon of
argF
and many other
Thermus
genes (with or without an SD box), sequences were found to be complementary to nucleotides 1392 to 1409 of
Thermus
16S rRNA, suggesting that an mRNA-rRNA base pairing in this region is important for correct translation initiation.
Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis
Authors: Sharma Susan , Davidson Amy L. 
Published: July 2002

ABSTRACT

The maltose transport system in
Escherichia coli
is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK
2
transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl
2
or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.
Genetic Analysis of Peptidoglycan Biosynthesis in Mycobacteria: Characterization of a
ddlA
Mutant of
Mycobacterium smegmatis
Authors: Belanger Aimee E. , Porter Joelle C. , Hatfull Graham F. 
Published: July 2002

ABSTRACT

A temperature-sensitive mutant of
Mycobacterium smegmatis
was characterized that contains a mutation in
ddlA
, the gene encoding
d
-alanine:
d
-alanine ligase. Enzymatic assays using recombinant proteins and
d
-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.
Two Regions of EpsL Involved in Species-Specific Protein-Protein Interactions with EpsE and EpsM of the General Secretion Pathway in
Vibrio cholerae
Authors: Sandkvist Maria , Keith Jerry M. , Bagdasarian Michael , Howard S. Peter 
Published: July 2002

ABSTRACT

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in
Vibrio cholerae
are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from
Aeromonas hydrophila
together with either EpsE or its
Aeromonas
homologue, ExeE, to complement the secretion defect in both
epsL
and
exeL
mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.
Three New NifA-Regulated Genes in the
Bradyrhizobium japonicum
Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization
Authors: Nienaber Andrea , Huber Alexander , Göttfert Michael , Hennecke Hauke , Fischer Hans-Martin 
Published: July 2002

ABSTRACT

The so-called symbiotic region of the
Bradyrhizobium japonicum
chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613–622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown
nifA
and
rpoN
mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized
nif
and
fix
gene clusters, we identified three new NifA-regulated genes that were named
nrgA
,
nrgB
, and
nrgC
(
nrg
stands for NifA-regulated gene). The latter two probably form an operon,
nrgBC
. The proteins encoded by
nrgC
and
nrgA
exhibited amino acid sequence similarity to bacterial hydroxylases and
N
-acetyltransferases, respectively. The product of
nrgB
showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational
lacZ
fusions revealed the presence of a functional −24/−12-type promoter upstream of
nrgA
and
nrgBC
and proved the NifA- and RpoN (ς
54
)-dependent transcription of the respective genes. Null mutations introduced into
nrgA
and
nrgBC
resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of
nrgA
and
nrgBC
further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.
Mutations in the
gerP
Locus of
Bacillus subtilis
and
Bacillus cereus
Affect Access of Germinants to Their Targets in Spores
Authors: Behravan Javad , Chirakkal Haridasan , Masson Anne , Moir Anne 
Published: July 2002

ABSTRACT

The
gerP1
transposon insertion mutation of
Bacillus cereus
is responsible for a defect in the germination response of spores to both
l
-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of
gerP
mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (
yisH-yisC
) can be found in the
Bacillus subtilis
genome sequence; null mutations in
yisD
and
yisF
, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in
B. cereus
, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a
gerP
mutant by the introduction of a
cotE
mutation, which renders the spore coats permeable to lysozyme. The
B. subtilis
operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the
Bacillus
spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.