bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth Phase Influences Complement Resistance ofBordetella pertussis
Authors: Barnes Michael G. , Weiss Alison A.
Published: July 2002

The BrkA protein ofBordetella pertussisinhibits killing by the antibody-dependent classical pathway of complement; however, susceptibility to complement can be highly variable. Log-phase bacteria grown in Stainer-Scholte (SS) broth plated on Bordet-Gengou (BG) agar were about 500 times more sensitive to killing by complement than stationary-phase SS-BG cultures. While always more susceptible to complement than the wild-type strain, a BrkA mutant displayed a similar growth phase variation in susceptibility to complement. Growth phase susceptibility to complement was also observed for a mutant constitutive for Bvg activation of BrkA, suggesting that modulation of virulence factor expression was not responsible for sensitivity to complement. Susceptibility was not due to differential antigenic expression, since serum adsorbed with complement-resistant, stationary-phase SS-BG cultures lacked bactericidal activity againstB. pertussisharvested at all times during the growth cycle. These results suggest that log-phase susceptibility to complement is not due to variable expression of BrkA or antigenic differences and may be an inherent property of rapidly growing cultures. Implications for vaccine development are discussed.
Identification and Characterization of a Nonimmunoglobulin Factor in Human Saliva That Inhibits
Streptococcus mutans
Glucosyltransferase
Authors: Jespersgaard Christina , Hajishengallis George , Russell Michael W. , Michalek Suzanne M.
Published: July 2002

Saliva contains an array of nonimmunoglobulin defense factors which are thought to contribute to the protection of the hard and soft tissue surfaces of the oral cavity by modulating microbial colonization and metabolism. Here we report the discovery of a putative innate defense factor in human saliva that inhibits the glucosyltransferase (GTF) of
Streptococcus mutans
, a virulence enzyme involved in oral colonization by this pathogen. The GTF-inhibiting factor (GIF) was initially identified as a nonimmunoglobulin salivary component that interfered with detection of antibodies to the glucan-binding region (GLU) of GTF by an enzyme-linked immunosorbent assay. This inhibitory activity was present in whole saliva and submandibular-sublingual saliva, but it was essentially absent from parotid saliva. GIF inhibited the recognition of
S. mutans
cell surface-associated GTF by specific antibodies but had no effect on antibodies to other cell surface antigens, suggesting that GIF specifically binds to GTF on
S. mutans.
GIF purified by size exclusion or affinity chromatography was used for biochemical and functional characterization. Analysis of GIF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high-molecular-weight glycoprotein after staining with Coomassie blue or Schiff's reagent. Heating and reduction with 2-mercaptoethanol of GIF resulted in the release of a ∼58-kDa protein that was identified as α-amylase by Western blotting using anti-α-amylase antibodies. GLU bound blotted α-amylase, suggesting that the latter molecule is the GLU-binding component of the GIF complex. The ability of GTF to synthesize extracellular glucans was inhibited by GIF but not by uncomplexed α-amylase or an unrelated high-molecular-weight glycoprotein. In conclusion, our findings demonstrate that in human saliva, there is a high-molecular-weight glycoprotein-α-amylase complex which is capable of inhibiting GTF and may contribute to control of
S. mutans
colonization in the oral cavity.
Fibronectin FacilitatesMycobacterium tuberculosisAttachment to Murine Alveolar Macrophages
Authors: Pasula Rajamouli , Wisniowski Paul , Martin William J.
Published: July 2002

Mycobacterium tuberculosisremains a major cause of pulmonary infection worldwide. Attachment ofM. tuberculosisorganisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bindM. tuberculosisorganisms and facilitates attachment ofM. tuberculosisto murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases125I-Fn binding toM. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding toM. tuberculosis. In the presence of exogenous Fn (10 μg/ml)M. tuberculosisattachment to AMs increased significantly from control levels (means ± standard errors of the means) of 11.5% ± 1.1% to 44.2% ± 4.2% (P< 0.05). Fn-enhanced attachment was significantly decreased from 44.2% ± 4.2% to 10.8% ± 1.2% (P< 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% ± 4.2% to 15.3% ± 1.2% (P< 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% ± 1.8%). These results suggest that Fn-mediated attachment ofM. tuberculosiscan occur through the binding of Fn to the AM via the CBD and toM. tuberculosisorganisms via the HBD.
Transmission Intensity Affects Both Antigen-Specific and Nonspecific T-Cell Proliferative Responses inLoa loaInfection
Authors: Akue Jean Paul , Devaney Eileen
Published: July 2002

T-cell proliferative responses were studied in two villages in Gabon with different levels ofLoa loatransmission. The first village (Okoumbi) had an annual transmission potential (ATP) of ≈9,000 infective larvae (L3)/person/year (high transmission village), while the second village (Ndjokaye) had an ATP of ≈1,000 L3/person/year (low transmission village). Proliferation and cytokine assays were performed on peripheral blood mononuclear cells (PBMC) from individuals aged 18 years and over using either mitogens (concanavalin A or phytohemagglutinin), antigens (purified protein derivative [PPD], irrelevant antigen), or soluble extracts of L3, microfilariae, or adultL. loa. PBMC from individuals in the low transmission village responded better to stimulation with adult antigen and to PPD than did PBMC from individuals in the high transmission village (P= 0.0031 andP= 0.0012, respectively). These data suggest that high levels of transmission ofL. loadepress both specific and nonspecific T-cell proliferative responses in infected humans.
Disruption of the
Candida albicans TPS2
Gene Encoding Trehalose-6-Phosphate Phosphatase Decreases Infectivity without Affecting Hypha Formation
Authors: Van Dijck Patrick , De Rop Larissa , Szlufcik Karolina , Van Ael Elke , Thevelein Johan M.
Published: July 2002

Deletion of trehalose-6-phosphate phosphatase, encoded by
TPS2
, in
Saccharomyces cerevisiae
results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in
Candida albicans
. We have cloned the
C. albicans TPS2
(
CaTPS2
) gene and constructed heterozygous and homozygous deletion strains. As in
S. cerevisiae
, complete inactivation of Tre6P phosphatase in
C. albicans
results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44°C. As opposed to inactivation of Tre6P synthase by deletion of
CaTPS1
, deletion of
CaTPS2
does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain.
CaTPS2
is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.
Purification and Characterization of Arginine Carboxypeptidase Produced by
Porphyromonas gingivalis
Authors: Masuda Kaname , Yoshioka Masami , Hinode Daisuke , Nakamura Ryo
Published: July 2002

Arginine carboxypeptidase was isolated from the cytoplasm of
Porphyromonas gingivalis
381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SW
XL
. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the
P. gingivalis
W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA,
o
-phenanthroline,
dl
-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu
2+
, Zn
2+
, and Cd
2+
. On the other hand, Co
2+
activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by
P. gingivalis
is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of
P. gingivalis
.
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

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

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

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

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

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

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

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

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

The 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

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

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

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

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

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

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

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.
Identification and Characterization of a Developmentally Regulated Protein, EshA, Required for Sporogenic Hyphal Branches in
Streptomyces griseus
Authors: Kwak Jangyul , McCue Lee Ann , Trczianka Kristen , Kendrick Kathleen E.
Published: July 2002

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in
Streptomyces
, we examined total proteins of
Streptomyces griseus
by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium,
Synechococcus
, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The
eshA
gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the
eshA
transcript depended on a developmental gene,
bldA
. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.
Flagellum-Independent Surface Migration of
Vibrio cholerae
and
Escherichia coli
Authors: Brown Igor I. , Häse Claudia C.
Published: July 2002

Surface translocation has been described in a large variety of microorganisms, including some gram-negative enteric bacteria. Here, we describe the novel observation of the flagellum-independent migration of
Vibrio cholerae
and
Escherichia coli
on semisolid surfaces with remarkable speeds. Important aspects of this motility are the form of inoculation, the medium composition, and the use of agarose rather than agar. Mutations in several known regulatory or surface structure proteins, such as ToxR, ToxT, TCP, and PilA, did not affect migration, whereas a defect in lipopolysaccharide biosynthesis prevented translocation. We propose that the observed surface migration is an active process, since heat, protease, or chloramphenicol treatments of the cells have strong negative effects on this phenotype. Furthermore, several
V. cholerae
strains strongly expressing the hemagglutinin/protease but not their isogenic
hap
-negative mutants, lacked the ability of surface motility, and the treatment of migrating strains with culture supernatants from
hap
strains but not
hap
-null strains prevented surface translocation.
Localization of a Germinant Receptor Protein (GerBA) to the Inner Membrane of
Bacillus subtilis
Spores
Authors: Paidhungat Madan , Setlow Peter
Published: July 2002

Dormant
Bacillus subtilis
spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the
gerA
family of operons, of which the
gerB
operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a
gerB
-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of
gerB
operon expression and with the putative receptor function of the proteins encoded by the
gerB
operon.
Mapping ofMycobacterium tuberculosis katGPromoters and Their Differential Expression in Infected Macrophages
Authors: Master Sharon , Zahrt Thomas C. , Song Jian , Deretic Vojo
Published: July 2002

Intracellular pathogenic bacteria, includingMycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant inM. tuberculosisis thekatGgene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence renderingM. tuberculosisexquisitely sensitive to this compound. In this context,katGexpression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH. Thus, it is important to delineate the regulation and expression ofkatG, as this not only can aid understanding of howM. tuberculosissurvives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of thekatGpromoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped thekatGmRNA 5′ ends and demonstrated that two promoters, P1furAand P1katG, control transcription ofkatG. ThefurAandkatGgenes are cotranscribed from P1furA. Both P1furAand P1katGpromoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide. Studies carried out using the transcriptional fusions P1furA-gfp, P1katG-gfp, and P1furA-P1katG-gfpconfirmed the existence of twokatGpromoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulentM. tuberculosisH37Rv. P1furAis induced early upon infection, and P1katGbecomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of thefurA-katGregion and indicate differential regulation in vivo of the twokatGpromoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.
The Histone Deacetylase GenesHDA1andRPD3Play Distinct Roles in Regulation of High-Frequency Phenotypic Switching inCandida albicans
Authors: Srikantha T. , Tsai L. , Daniels K. , Klar A. J. S. , Soll D. R.
Published: July 2002

Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2,andHOS3) have been cloned fromCandida albicansand characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylasesHDA1andRPD3is down-regulated in the opaque phase of the white-opaque transition in strain WO-1.HOS3is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase.HDA1andRPD3were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion ofHDA1resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion ofRPD3resulted in an increase in the frequency of switching in both directions. Deletion ofHDA1resulted in reduced white-phase-specific expression of theEFG13.2-kb transcript,but had no significant effect on white-phase-specific expression ofWH11or opaque-phase-specific expression ofOP4, SAP1,andSAP3.Deletion ofRPD3resulted in reduced opaque-phase-specific expression ofOP4, SAP1,andSAP3and a slight reduction of white-phase-specific expression ofWH11and 3.2-kbEFG1. Deletion of neitherHDA1norRPD3affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein geneMCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genesHDA1andRPD3play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.
Comparison of Δ
relA
Strains of
Escherichia coli
and
Salmonella enterica
Serovar Typhimurium Suggests a Role for ppGpp in Attenuation Regulation of Branched-Chain Amino Acid Biosynthesis
Authors: Tedin K. , Norel F.
Published: July 2002

The growth recovery of
Escherichia coli
K-12 and
Salmonella enterica
serovar Typhimurium Δ
relA
mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type
E. coli
K-12 and
S. enterica
serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (
ilvGM
) and III (
ilvIH
) isozymes, respectively, Δ
relA
derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the Δ
relA
strains. In the
E. coli
K-12 MG1655 Δ
relA
background, correction of the preexisting
rph
-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery.
S. enterica
serovar Typhimurium LT2 Δ
relA
strains were fully complemented by elevated basal ppGpp levels in an
S. enterica
serovar Typhimurium LT2 Δ
relA spoT1
mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by
relA
-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the
E. coli
K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.
Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division
Authors: Mukherjee Amit , Saez Cristian , Lutkenhaus Joe
Published: July 2002

FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the
ftsZ2
mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.
Aminopeptidases A, B, and N and Dipeptidase D Are the Four Cysteinylglycinases of
Escherichia coli
K-12
Authors: Suzuki Hideyuki , Kamatani Sachiko , Kim Eun-Soo , Kumagai Hidehiko
Published: July 2002

Aminopeptidases A, B, and N and dipeptidase D, with broad substrate specificity, are the four cysteinylglycinases of
Escherichia coli
K-12, and there is no peptidase specific for the cleavage of cysteinylglycine.
Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1
Authors: Szpirer Cédric Y. , Faelen Michel , Couturier Martine
Published: July 2002

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium
Bordetella bronchiseptica
(R. Antoine and C. Locht, Mol. Microbiol. 6:1785–1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the
mob
gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and
mob
gene regulation. We show that the Mob protein is a relaxase, and we located the
nic
site position in vitro. After sequence alignment, the position of the
nic
site of pBBR1 corresponds with those of the nick sites of the
Bacteroides
mobilizable transposon Tn
4555
and the streptococcal plasmid pMV158. The
oriT
of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.