bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTSalmonella 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

ABSTRACT

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

ABSTRACT

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.