bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Chemokines Are Induced byMycobacterium tuberculosisand Inhibit Its Growth
Authors: Saukkonen Jussi J. , Bazydlo Beth , Thomas Michael , Strieter Robert M. , Keane Joseph , Kornfeld Hardy
Published: July 2002

Chemokines (CK) are potent leukocyte activators and chemoattractants and aid in granuloma formation, functions critical for the immune response toMycobacterium tuberculosis. We hypothesized that infection of alveolar macrophages (AM) with different strains ofM. tuberculosiselicits distinct profiles of CK, which could be altered by human immunodeficiency virus (HIV) infection. RANTES, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β were the major β-CK produced in response toM. tuberculosisinfection. VirulentM. tuberculosis(H37Rv) induced significantly less MIP-1α than did the avirulent strain (H37Ra), while MIP-1β and RANTES production was comparable for both strains. MIP-1α and MIP-1β were induced by the membrane, but not cytosolic, fraction ofM. tuberculosis.M. tuberculosis-induced CK secretion was partly dependent on tumor necrosis factor alpha (TNF-α). AM from HIV-infected individuals produced less TNF-α and MIP-1β than did normal AM in response to eitherM. tuberculosisstrain. We tested the functional significance of decreased β-CK secretion by examining the ability of β-CK to suppress intracellular growth ofM. tuberculosis. MIP-1β and RANTES suppressed intracellular growth ofM. tuberculosistwo- to threefold, a novel finding. Thus, β-CK contribute to the innate immune response toM. tuberculosisinfection, and their diminution may promote the intracellular survival ofM. tuberculosis.
Proteolysis of CD14 on Human Gingival Fibroblasts by Arginine-Specific Cysteine Proteinases fromPorphyromonas gingivalisLeading to Down-Regulation of Lipopolysaccharide-Induced Interleukin-8 Production
Authors: Tada Hiroyuki , Sugawara Shunji , Nemoto Eiji , Takahashi Nobuhiro , Imamura Takahisa , Potempa Jan , Travis James , Shimauchi Hidetoshi , Takada Haruhiko
Published: July 2002

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathicPorphyromonas gingivaliscleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.
The H
2
Sensor of
Ralstonia eutropha
Is a Member of the Subclass of Regulatory [NiFe] Hydrogenases
Authors: Kleihues Laura , Lenz Oliver , Bernhard Michael , Buhrke Thorsten , Friedrich Bärbel
Published: July 2002

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium
Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H
2
, which is available in low amounts in aerobic environments. H
2
sensing in
R. eutropha
is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H
2
sensor of
R. eutropha
is a cytoplasmic protein. Although capable of H
2
oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for
R. eutropha
provided the basis for identifying the H
2
sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231–235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H
2
sensor is nickel dependent. The results suggest that H
2
sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H
2
binding or subsequent H
2
oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H
2
-sensing hydrogenase of
R. eutropha
, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H
2
sensors represents a novel subclass of [NiFe] hydrogenases denoted as the “regulatory hydrogenases.”
Contribution of Cysteine Desulfurase (NifS Protein) to the Biotin Synthase Reaction of
Escherichia coli
Authors: Kiyasu Tatsuya , Asakura Akira , Nagahashi Yoshie , Hoshino Tatsuo
Published: July 2002

The contribution of cysteine desulfurase, the NifS protein of
Klebsiella pneumoniae
and the IscS protein of
Escherichia coli
, to the biotin synthase reaction was investigated in in vitro and in vivo reaction systems with
E. coli
. When the
nifS
and
nifU
genes of
K. pneumoniae
were coexpressed in
E. coli
, NifS and NifU proteins in complex (NifU/S complex) and NifU monomer forms were observed. Both the NifU/S complex and the NifU monomer stimulated the biotin synthase reaction in the presence of
l
-cysteine in an in vitro reaction system. The NifU/S complex enhanced the production of biotin from dethiobiotin by the cells growing in an in vivo reaction system. Moreover, the IscS protein of
E. coli
stimulated the biotin synthase reaction in the presence of
l
-cysteine in the cell-free system. These results strongly suggest that cysteine desulfurase participates in the biotin synthase reaction, probably by supplying sulfur to the iron-sulfur cluster of biotin synthase.
Mutational Analysis of the
sbo-alb
Locus of
Bacillus subtilis
: Identification of Genes Required for Subtilosin Production and Immunity
Authors: Zheng Guolu , Hehn Robin , Zuber Peter
Published: July 2002

The
Bacillus subtilis
168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against
Listeria monocytogenes
. Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene
sboA
and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process. The genes of the
sbo-alb
operon are believed to function in the synthesis and maturation of subtilosin. Nonpolar mutations introduced into each of the
alb
genes resulted in loss or reduction of subtilosin production.
sboA
,
albA
, and
albF
mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin. Mutations in
albB
, -
C
, and -
D
resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions. A new gene,
sboX
, encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of
sboA
. Construction of an
sboX-lacZ
translational fusion and analysis of its expression indicate that
sboX
is induced in stationary phase of anaerobic cultures of JH642. An in-frame deletion of the
sboX
coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin. The results of this investigation show that the
sbo-alb
genes are required for the mechanisms of subtilosin synthesis and immunity.
Green Fluorescent Protein Functions as a Reporter for Protein Localization in
Escherichia coli
Authors: Feilmeier Bradley J. , Iseminger Ginger , Schroeder Diane , Webber Hannali , Phillips Gregory J.
Published: July 2002

The use of green fluorescent protein (GFP) as a reporter for protein localization in
Escherichia coli
was explored by creating gene fusions between
malE
(encoding maltose-binding protein [MBP]) and a variant of
gfp
optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different
sec
mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the
sec
-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate
malE
signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported β-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.
Analysis of the Function of a Putative 2,3-Diphosphoglyceric Acid-Dependent Phosphoglycerate Mutase from
Bacillus subtilis
Authors: Pearson Claire L. , Loshon Charles A. , Pedersen Lotte B. , Setlow Barbara , Setlow Peter
Published: July 2002

A
Bacillus subtilis
gene termed
yhfR
encodes the only
B. subtilis
protein with significant sequence similarity to 2,3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM). This gene is expressed at a low level during growth and sporulation, but deletion of
yhfR
had no effect on growth, sporulation, or spore germination and outgrowth. YhfR was expressed in and partially purified from
Escherichia coli
but had little if any PGM activity and gave no detectable PGM activity in
B. subtilis
. These data indicate that
B. subtilis
does not require YhfR and most likely does not require a dPGM.
Tissue-Specific Gene Expression Identifies a Gene in the Lysogenic Phage Gifsy-1 That Affects
Salmonella enterica
Serovar Typhimurium Survival in Peyer's Patches
Authors: Stanley Theresa L. , Ellermeier Craig D. , Slauch James M.
Published: July 2002

In vivo expression technology was used to identify
Salmonella enterica
serovar Typhimurium genes that are transcriptionally induced when the bacteria colonize the small intestines of mice. These genes were subsequently screened for those that are transcriptionally inactive during the systemic stages of disease. This procedure identified
gipA
, a gene that is specifically induced in the small intestine of the animal. The
gipA
gene is carried on the lambdoid phage Gifsy-1. Consistent with the expression profile, the sole defect conferred by a
gipA
null mutation is in growth or survival in a Peyer's patch. The
gipA
strain is wild type in its ability to initially colonize the small intestine and invade the intestinal epithelium. The mutant also survives and propagates at wild-type levels during the systemic stages of disease. The
gipA
open reading frame is homologous to a family of putative insertion sequence elements, although our evidence shows that transposition is not required for
gipA
function in the Peyer's patch. These results suggest that the bacteria sense and respond to the particular environment of the Peyer's patch, a critical site for the replication of
Salmonella
serovar Typhimurium.
Regulation of Expression of the
yiaKLMNOPQRS
Operon for Carbohydrate Utilization in
Escherichia coli
: Involvement of the Main Transcriptional Factors
Authors: Ibañez Ester , Campos Evangelina , Baldoma Laura , Aguilar Juan , Badia Josefa
Published: July 2002

The
yiaKLMNOPQRS
(
yiaK-S
) gene cluster of
Escherichia coli
is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate
l
-xylulose. Transcription of
yiaK-S
as a single message from the unique promoter found upstream of
yiaK
is proven in this study. The 5′ end has been located at 60 bp upstream from the ATG. Expression of the
yiaK-S
operon is controlled in the wild-type strain by a repressor encoded by
yiaJ
. No inducer molecule of the
yiaK-S
operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. The
lacZ
transcriptional fusions in the genetic background of the mutant strain revealed that
yiaK-S
is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from −50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. The
lacZ
transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at −106, is essential for
yiaK-S
expression. The 5′ end of the
yiaJ
gene was determined, and its promoter region was found to overlap with the divergent
yiaK-S
promoter. Expression of
yiaJ
is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of the
yiaK-S
promoter.
Spy1, a Histidine-Containing Phosphotransfer Signaling Protein, Regulates the Fission Yeast Cell Cycle through the Mcs4 Response Regulator
Authors: Aoyama Keisuke , Mitsubayashi Yasunori , Aiba Hirofumi , Mizuno Takeshi
Published: July 2002

Common histidine-to-aspartate (His-to-Asp) phosphorelay signaling systems involve three types of signaling components: a sensor His kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) protein. In the fission yeast
Schizosaccharomyces pombe
, two response regulators, Mcs4 and Prr1, have been identified recently, and it was shown that they are involved in the signal transduction implicated in stress responses. Furthermore, Mcs4 appears to be involved in mitotic cell-cycle control. However, neither the HPt phosphotransmitter nor His kinase has been characterized in
S. pombe
. In this study, we identified a gene encoding an HPt phosphotransmitter, named Spy1 (
S. pombe
YPD1-like protein). The

spy1
+

gene showed an ability to complement a mutational lesion of the
Saccharomyces cerevisiae YPD1
gene, which is involved in an osmosensing signal transduction. The result from yeast two-hybrid analysis indicated that Spy1 interacts with Mcs4. To gain insight into the function of Spy1, a series of genetic analyses were conducted. The results provided evidence that Spy1, together with Mcs4, plays a role in regulation of the G
2
/M cell cycle progression. Spy1-deficient cells appear to be precocious in the entry to M phase. In the proposed model, Spy1 modulates Mcs4 in a negative manner, presumably through a direct His-to-Asp phosphorelay, operating upstream of the Sty1 mitogen-activated protein kinase cascade.
A Scaffoldin of the
Bacteroides cellulosolvens
Cellulosome That Contains 11 Type II Cohesins
Authors: Ding Shi-You , Bayer Edward A. , Steiner David , Shoham Yuval , Lamed Raphael
Published: July 2002

A cellulosomal scaffoldin gene, termed
cipBc
, was identified and sequenced from the mesophilic cellulolytic anaerobe
Bacteroides cellulosolvens
. The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437. CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain. Its CBD belongs to family IIIb, like that of CipV from
Acetivibrio cellulolyticus
but unlike the family IIIa CBDs of other clostridial scaffoldins. In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function. The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence. One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment. By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II. This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component. The results attest to the emerging diversity of cellulosomes and their component sequences in nature.
The
orf162b
Sequence of
Rhodobacter capsulatus
Encodes a Protein Required for Optimal Levels of Photosynthetic Pigment-Protein Complexes
Authors: Aklujkar Muktak , Harmer Andrea L. , Prince Roger C. , Beatty J. Thomas
Published: July 2002

The
orf162b
sequence, the second open reading frame 3′ of the reaction center (RC) H protein gene
puhA
in the
Rhodobacter capsulatus
photosynthesis gene cluster, is shown to be transcribed from a promoter located 5′ of
puhA
. A nonpolar mutation of
orf162b
was generated by replacing most of the coding region with an antibiotic resistance cartridge. Although the mutant strain initiated rapid photosynthetic growth, growth slowed progressively and cultures often entered a pseudostationary phase. The amounts of the RC and light harvesting complex I (LHI) in cells obtained from such photosynthetic cultures were abnormally low, but these deficiencies were less severe when the mutant was grown to a pseudostationary phase induced by low aeration in the absence of illumination. The
orf162b
mutation did not significantly affect the expression of a
pufB
::
lacZ
translationally in-frame gene fusion under the control of the
puf
promoter, indicating normal transcription and translation of RC and LHI genes. Spontaneous secondary mutations in the strain with the
orf162b
disruption resulted in a bypass of the photosynthetic growth retardation and reduced the level of light harvesting complex II. These results and the presence of sequences similar to
orf162b
in other species indicate that the Orf162b protein is required for normal levels of the photosynthetic apparatus in purple photosynthetic bacteria.
No Genetic Barriers between
Salmonella enterica
Serovar Typhimurium and
Escherichia coli
in SOS-Induced Mismatch Repair-Deficient Cells
Authors: Matic Ivan , Taddei François , Radman Miroslav
Published: July 2002

Conjugational crosses trigger SOS induction in
Escherichia coli
F
−
cells mated with
Salmonella enterica
serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera.
Control of Lactose Transport, β-Galactosidase Activity, and Glycolysis by CcpA in
Streptococcus thermophilus
: Evidence for Carbon Catabolite Repression by a Non-Phosphoenolpyruvate-Dependent Phosphotransferase System Sugar
Authors: van den Bogaard Patrick T. C. , Kleerebezem Michiel , Kuipers Oscar P. , de Vos Willem M.
Published: July 2002

Streptococcus thermophilus
, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, β-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (
cre
) was identified in the promoter of the
lacSZ
operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of
lacSZ
transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted
ccpA
gene. Moreover, a
ccpA
disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the
ldh
gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a
ccpA
disruption mutant. We propose that CcpA in
S. thermophilus
acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria,
S. thermophilus
possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.
Geranylgeranyltransferase I of
Candida albicans
: Null Mutants or Enzyme Inhibitors Produce Unexpected Phenotypes
Authors: Kelly Rosemarie , Card Deborah , Register Elizabeth , Mazur Paul , Kelly Theresa , Tanaka Ken-Ichi , Onishi Janet , Williamson Joanne M. , Fan Hongxia , Satoh Toshihiko , Kurtz Myra
Published: July 2002

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyl diphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine; a, usually aliphatic amino acid; X, usually L). The α and β subunits of GGTase I from
Saccharomyces cerevisiae
are encoded by
RAM2
and
CDC43
, respectively, and each is essential for viability. We are evaluating GGTase I as a potential target for antimycotic therapy of the related yeast,
Candida albicans
, which is the major human pathogen for disseminated fungal infections. Recently we cloned
CaCDC43
, the
C. albicans
homolog of
S. cerevisiae CDC43
. To study its role in
C. albicans
, both alleles were sequentially disrupted in strain CAI4. Null
Cacdc43
mutants were viable despite the lack of detectable GGTase I activity but were morphologically abnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from the membranous fraction to the cytosolic fraction in the
cdc43
mutants, and levels of these two proteins were elevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitro but that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution and quantity of the substrate. The lethality of an
S. cerevisiae cdc43
mutant can be suppressed by simultaneous overexpression of
RHO1
and
CDC42
on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017, 1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs by farnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of
C. albicans
can be prenylated by FTase when GGTase I is absent or limiting and that elevation of these two substrates enables them to compete with FTase substrates for prenylation and thus allows sustained growth.
Complex Function for SicA, aSalmonella entericaSerovar Typhimurium Type III Secretion-Associated Chaperone
Authors: Tucker Stephanie C. , Galán Jorge E.
Published: July 2002

Salmonella entericaencodes a type III secretion system within a pathogenicity island located at centisome 63 that is essential for virulence. All type III secretion systems require the function of a family of low-molecular-weight proteins that aid the secretion process by acting as partitioning factors and/or secretion pilots. One such protein is SicA, which is encoded immediately upstream of the type III secreted proteins SipB and SipC. We found that the absence of SicA results in the degradation of both SipB and SipC. Interestingly, in the absence of SipC, SipB was not only stable but also secreted at wild-type levels in asicAmutant background, indicating that SicA is not required for SipB secretion. We also found that SicA is capable of binding both SipB and SipC. These results are consistent with a SicA role as a partitioning factor for SipB and SipC, thereby preventing their premature association and degradation. We also found that introduction of asicAnull mutation results in the lack of expression of SopE, another type III-secreted protein. Such an effect was shown to be transcriptional. Introduction of a loss-of-functionsipCmutation into thesicAmutant background rescuedsopEexpression. These results indicate that the effect ofsicAonsopEexpression is indirect and most likely exerted through a regulatory factor(s) partitioned by SicA from SipC. These studies therefore describe a surprisingly complex function for theSalmonella entericatype III secretion-associated chaperone SicA.
Initiation Factor 2 of
Myxococcus xanthus
, a Large Version of Prokaryotic Translation Initiation Factor 2
Authors: Tiennault-Desbordes Emmanuelle , Cenatiempo Yves , Laalami Soumaya
Published: July 2002

We have isolated the structural gene for translation initiation factor IF2 (
infB
) from the myxobacterium
Myxococcus xanthus
. The gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its G domain and C terminus to the equivalent regions of IF2 from
Escherichia coli
. The protein cross-reacts with antibodies raised against
E. coli
IF2 and was able to complement an
E. coli infB
mutant. The
M. xanthus
protein is the largest IF2 known to date. This is essentially due to a longer N-terminal region made up of two characteristic domains. The first comprises a 188-amino-acid sequence consisting essentially of alanine, proline, valine, and glutamic acid residues, similar to the APE domain observed in
Stigmatella aurantiaca
IF2. The second is unique to
M. xanthus
IF2, is located between the APE sequence and the GTP binding domain, and consists exclusively of glycine, proline, and arginine residues.
Partial Characterization of an Enzyme Fraction with Protease Activity Which Converts the Spore Peptidoglycan Hydrolase (SleC) Precursor to an Active Enzyme during Germination of
Clostridium perfringens
S40 Spores and Analysis of a Gene Cluster Involved in the Activity
Authors: Shimamoto Seiko , Moriyama Ryuichi , Sugimoto Kazuhiro , Miyata Shigeru , Makino Shio
Published: July 2002

A spore cortex-lytic enzyme of
Clostridium perfringens
S40 which is encoded by
sleC
is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55°C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl
2
indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of
cspA, cspB
, and
cspC
, which are positioned in a tandem array just upstream of the 5′ end of
sleC
. The amino acid sequences deduced from the nucleotide sequences of the
csp
genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.
Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant
Pseudomonas putida
DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks
Authors: Segura Ana , Duque Estrella , Hurtado Ana , Ramos Juan L.
Published: July 2002

Pseudomonas putida
DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn
5-′phoA
-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn
5-′phoA
-Km was inserted within the
fliP
gene, which is part of the
fliLMNOPQRflhBA
cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the
P. putida fliP
mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An
flhB
knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the
fliL
gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in
P. putida
DOT-T1E to the periplasm.
Computation-Directed Identification of OxyR DNA Binding Sites in
Escherichia coli
Authors: Zheng Ming , Wang Xunde , Doan Bernard , Lewis Karen A. , Schneider Thomas D. , Storz Gisela
Published: July 2002

A computational search was carried out to identify additional targets for the
Escherichia coli
OxyR transcription factor. This approach predicted OxyR binding sites upstream of
dsbG
, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of
fhuF
, encoding a protein required for iron uptake; and within
yfdI
. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the
dsbG
gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed
ahpC
gene. Although the new binding site was near
dsbG
, Northern blotting and primer extension assays showed that OxyR binding to the
dsbG
-proximal site led to the induction of a second
ahpCF
transcript, while OxyR binding to the
ahpCF
-proximal site leads to the induction of both
dsbG
and
ahpC
transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of the
fhuF
gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the
fhuF
promoter. Interestingly, the two OxyR sites in the
fhuF
promoter overlapped with two regions bound by the Fur repressor. Expression analysis revealed that
fhuF
was repressed by hydrogen peroxide in an OxyR-dependent manner. Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of
yfdI
. These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the
E. coli
genome.
Genetic and Physiologic Analysis of the
groE
Operon and Role of the HrcA Repressor in Stress Gene Regulation and Acid Tolerance in
Streptococcus mutans
Authors: Lemos José A. C. , Chen Yi-Ywan M. , Burne Robert A.
Published: July 2002

Our working hypothesis is that the major molecular chaperones DnaK and GroE play central roles in the ability of oral bacteria to cope with the rapid and frequent stresses encountered in oral biofilms, such as acidification and nutrient limitation. Previously, our laboratory partially characterized the
dnaK
operon of
Streptococcus mutans
(
hrcA
-
grpE
-
dnaK
) and demonstrated that
dnaK
is up-regulated in response to acid shock and sustained acidification (G. C. Jayaraman, J. E. Penders, and R. A. Burne, Mol. Microbiol. 25:329–341, 1997). Here, we show that the
groESL
genes of
S. mutans
constitute an operon that is expressed from a stress-inducible ς
A
-type promoter located immediately upstream of a CIRCE element. GroEL protein and mRNA levels were elevated in cells exposed to a variety of stresses, including acid shock. A nonpolar insertion into
hrcA
was created and used to demonstrate that HrcA negatively regulates the expression of the
groEL
and
dnaK
operons. The SM11 mutant, which had constitutively high levels of GroESL and roughly 50% of the DnaK protein found in the wild-type strain, was more sensitive to acid killing and could not lower the pH as effectively as the parent. The acid-sensitive phenotype of SM11 was, at least in part, attributable to lower F
1
F
0
-ATPase activity. A minimum of 10 proteins, in addition to GroES-EL, were found to be up-regulated in SM11. The data clearly indicate that HrcA plays a key role in the regulation of chaperone expression in
S. mutans
and that changes in the levels of the chaperones profoundly influence acid tolerance.
Hemin Reconstitutes Proton Extrusion in an H

+

-ATPase-Negative Mutant of
Lactococcus lactis
Authors: Blank Lars M. , Koebmann Brian J. , Michelsen Ole , Nielsen Lars K. , Jensen Peter R.
Published: July 2002

H
+
-ATPase is considered essential for growth of
Lactococcus lactis.
However, media containing hemin restored the aerobic growth of an H
+
-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown
L. lactis
cells are capable of coupling NADH oxidation to proton translocation.
Conversion of Pipecolic Acid into Lysine in
Penicillium chrysogenum
Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the
lys7
Gene Encoding Saccharopine Reductase
Authors: Naranjo Leopoldo , de Valmaseda Eva Martin , Bañuelos Oscar , Lopez Pilar , Riaño Jorge , Casqueiro Javier , Martin Juan F.
Published: July 2002

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In
Penicillium chrysogenum
pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the
lys1
,
lys2
, or
lys3
genes allowing a slow growth of these auxotrophs. We have isolated two
P. chrysogenum
mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with α-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named
lys7
) revealed that it encoded a protein with high similarity to the saccharopine reductase from
Neurospora crassa
,
Magnaporthe grisea
,
Saccharomyces cerevisiae
, and
Schizosaccharomyces pombe
. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that
lys7
encodes a functional saccharopine reductase. Our data suggest that in
P. chrysogenum
the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.
Nitrogen-Regulated Group 2 Sigma Factor from
Synechocystis
sp. Strain PCC 6803 Involved in Survival under Nitrogen Stress
Authors: Muro-Pastor Alicia Marı́a , Herrero Antonia , Flores Enrique
Published: July 2002

The expression of
sll1689
, an open reading frame from the cyanobacterium
Synechocystis
sp. strain PCC 6803 putatively encoding a member of the ς
70
family of sigma factors, appears to be regulated by the nitrogen control transcription factor NtcA. Disruption of
sll1689
had no noticeable effect on exponential growth, identifying its product as a member of the group 2, nonessential class of ς
70
-like sigma factors; however, this disruption decreased the viability of the cells after long periods of nitrogen starvation. We have named this gene
rpoD2
-
V
. The expression of
glnN
, encoding a type III glutamine synthetase, was impaired in strains bearing an inactivated copy of the
rpoD2
-
V
gene.
Comparative Specificities of Two Evolutionarily Divergent Hydrolases Involved in Microbial Degradation of Polychlorinated Biphenyls
Authors: Seah Stephen Y. K. , Labbé Geneviève , Kaschabek Stefan R. , Reifenrath Frank , Reineke Walter , Eltis Lindsay D.
Published: July 2002

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs) by
Burkholderia
sp. strain LB400 (S. Y. K. Seah, G. Labbé, S. Nerdinger, M. Johnson, V. Snieckus, and L. D. Eltis, J. Biol. Chem. 275:15701–15708, 2000). To determine whether this is also true in divergent biphenyl degraders, the homologous hydrolase of
Rhodococcus globerulus
P6, BphD
P6
, was hyperexpressed, purified to apparent homogeneity, and studied by steady-state kinetics. BphD
P6
hydrolyzed HOPDA with a
k
cat
/

K
m

of 1.62 (± 0.03) × 10
7
M
−1
s
−1
(100 mM phosphate [pH 7.5], 25°C), which is within 70% of that of BphD
LB400
. BphD
P6
was also similar to BphD
LB400
in that it catalyzed the hydrolysis of HOPDAs bearing chloro substituents on the phenyl moiety at least 25 times more specifically than those bearing chloro substituents on the dienoate moiety. However, the rhodococcal enzyme was significantly more specific for 9-Cl and 10-Cl HOPDAs, catalyzing the hydrolysis of 9-Cl, 10-Cl, and 9,10-diCl HOPDAs two- to threefold respectively, more specifically than HOPDA. Moreover, 4-Cl HOPDA competitively inhibited BphD
P6
more effectively than 3-Cl HOPDA, which is the inverse of what was observed in BphD
LB400
. These results demonstrate that BphD is a key determinant in the aerobic transformation of PCBs by divergent biphenyl degraders, but that there exists significant diversity in the specificity of these biphenyl hydrolases.
Dissimilatory Sulfite Reductase (Desulfoviridin) of the Taurine-Degrading, Non-Sulfate-Reducing Bacterium
Bilophila wadsworthia
RZATAU Contains a Fused DsrB-DsrD Subunit
Authors: Laue Heike , Friedrich Michael , Ruff Jürgen , Cook Alasdair M.
Published: July 2002

A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium
Bilophila wadsworthia
RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as an α
2
β
2
γ

n

(
n
≥ 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the α and β subunits (
dsrA
and
dsrB
, respectively), which probably constitute one operon.
dsrA
and
dsrB
encode polypeptides of 49 (α) and 54 kDa (β) which show significant similarities to the homologous subunits of other DSRs. The
dsrB
gene product of
B. wadsworthia
is apparently a fusion protein of
dsrB
and
dsrD
. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in
B. wadsworthia
. A phylogenetic analysis using the available Dsr sequences revealed that
B. wadsworthia
grouped with its closest 16S rDNA relative
Desulfovibrio desulfuricans
Essex 6.
The
miaA
Mutator Phenotype of
Escherichia coli
K-12 Requires Recombination Functions
Authors: Zhao Jingyong , Leung Hon-Chiu Eastwood , Winkler Malcolm E.
Published: July 2002

miaA
mutants, which contain A-37 instead of the ms
2
i
6
A-37 hypermodification in their tRNA, show a moderate mutator phenotype leading to increased GC→TA transversion. We show that the
miaA
mutator phenotype is dependent on recombination functions similar to, but not exactly the same as, those required for translation stress-induced mutagenesis.
Global Genomic Analysis of AlgU (σ
E
)-Dependent Promoters (Sigmulon) in
Pseudomonas aeruginosa
and Implications for Inflammatory Processes in Cystic Fibrosis
Authors: Firoved Aaron M. , Boucher J. Cliff , Deretic Vojo
Published: July 2002

The conversion of
Pseudomonas aeruginosa
to the mucoid phenotype coincides with the establishment of chronic respiratory infections in cystic fibrosis (CF). A major pathway of conversion to mucoidy in clinical strains of
P. aeruginosa
is dependent upon activation of the alternative sigma factor AlgU (
P. aeruginosa
σ
E
). Here we initiated studies of AlgU-dependent global expression patterns in
P. aeruginosa
in order to assess whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are turned on during conversion to mucoidy. Using genomic information and the consensus AlgU promoter sequence, we identified 35 potential AlgU (σ
E
) promoter sites on the
P. aeruginosa
chromosome. Each candidate promoter was individually tested by reverse transcription and mRNA 5′-end mapping using RNA isolated from
algU
+
and
algU
::Tc
r
mutant cells. A total of 10 new AlgU-dependent promoters were identified, and the corresponding mRNA start sites were mapped. Two of the 10 newly identified AlgU promoters were upstream of predicted lipoprotein genes. Since bacterial lipoproteins have been implicated as inducers of inflammatory pathways, we tested whether lipopeptides corresponding to the products of the newly identified AlgU-dependent lipoprotein genes,
lptA
and
lptB
, had proinflammatory activity. In human peripheral blood monocyte-derived macrophages the peptides caused production of interleukin-8, a proinflammatory chemokine typically present at excessively high levels in the CF lung. Our studies show how genomic information can be used to uncover on a global scale the genes controlled by a given σ factor (collectively termed here sigmulon) using conventional molecular tools. In addition, our data suggest the existence of a previously unknown connection between conversion to mucoidy and expression of lipoproteins with potential proinflammatory activity. This link may be of significance for infections and inflammatory processes in CF.
Further Evidence that a Cell Wall Precursor [C
55
-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction
Authors: Ruzin Alexey , Severin Anatoly , Ritacco Frank , Tabei Keiko , Singh Guy , Bradford Patricia A. , Siegel Marshall M. , Projan Steven J. , Shlaes David M.
Published: July 2002

Previous studies suggested that a Gly-containing branch of cell wall precursor [C
55
-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in
Staphylococcus aureus.
To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of
Streptomyces
spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.
Functional Measurement of Hepatitis C Virus Core-Specific CD8+T-Cell Responses in the Livers or Peripheral Blood of Patients by Using Autologous Peripheral Blood Mononuclear Cells as Targets or Stimulators
Authors: Fang Shih-Hua , Chiang Bor-Luen , Wu Mei-Hua , Iba Hideo , Lai Ming-Yang , Yang Pei-Ming , Chen Ding-Shinn , Hwang Lih-Hwa
Published: July 2002

As is widely recognized, CD8+cytotoxic T lymphocytes (CTLs) play a crucial role in hepatitis C virus (HCV) infection, both in pathogenesis of liver injury and in clearing the virus. CTL studies with HCV-infected patients have been difficult because of the relatively low frequency of CTL precursors in the peripheral blood and because the targeted epitopes vary depending on the human leukocyte antigen (HLA) types of the individuals. This study attempts to overcome these problems by assessing the feasibility of using autologous peripheral blood mononuclear cells (PBMCs) expressing viral antigens as stimulators or targets in order to monitor the CTL responses. Primary PBMCs were transduced using a retroviral vector pseudotyped with a vesicular stomatitis virus G glycoprotein expressing the HCV core gene. Additionally, the vector-transduced PBMCs were used as targets of CTL assays to measure the HCV core-specific CTL activities from the liver-infiltrating lymphocytes of six different HLA-type patients with chronic HCV infection. The core-expressing PBMCs also served as stimulators, allowing us to measure core-specific CD8+T-cell responses by intracellular gamma interferon staining of the peripheral blood of hepatitis C patients who had received treatment with alpha interferon plus ribavirin. This approach provides an efficient means of measuring antigen-specific CTL responses without HLA constraints.
Rotavirus Genotypes P[4]G9, P[6]G9, and P[8]G9 in Hospitalized Children with Acute Gastroenteritis in Rio de Janeiro, Brazil
Authors: Araújo I. T. , Ferreira M. S. R. , Fialho A. M. , Assis R. M. , Cruz C. M. , Rocha M. , Leite J. P. G.
Published: July 2002

Fifty-three rotavirus-positive fecal specimens from children with diarrhea admitted to a Rio de Janeiro, Brazil, children's hospital between January 1997 and December 1998 were characterized for P and G types by using reverse transcription-PCR. Genotype P[4]G2 accounted for 21% of isolates, while uncommon genotypes P[8]G9, P[6]G9, and P[4]G9 accounted for 13% of the isolates.
LightCycler Multiplex PCR for the Laboratory Diagnosis of Common Viral Infections of the Central Nervous System
Authors: Read Steven J. , Mitchell Joanne L. , Fink Colin G.
Published: July 2002

A conventional multiplex PCR assay that detects herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus, and enteroviruses for the diagnosis of central nervous system infections was modified to be performed using the LightCycler system. The sensitivity of detection of each of the viruses using the LightCycler assay was compared to that of the conventional assay using external quality assessment material. The assays had equivalent sensitivities, but the LightCycler assay was more rapid, reduced the risk of contamination, and used an amplicon detection format that demonstrated greater discrimination than a gel electrophoresis method.