bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deletion of New Covalently Linked Cell Wall Glycoproteins Alters the Electrophoretic Mobility of Phosphorylated Wall Components of
Saccharomyces cerevisiae
Authors: Mrsa Vladimir , Ecker Margit , Strahl-Bolsinger Sabine , Nimtz Manfred , Lehle Ludwig , Tanner Widmar 
Published: December 2019

ABSTRACT

The incorporation of radioactive orthophosphate into the cell walls of
Saccharomyces cerevisiae
was studied.
33
P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N -glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368–3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by
DAN1
[also called
TIR3
]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154–2162, 1997). In
ccw12
,
ccw13
, or
ccw14
single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the
33
P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of
CCW12
results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the
ccw12
mutant shows a decrease in mating efficiency and a defect in agglutination.
Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in
Streptococcus gordonii
Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties
Authors: McNab Roderick , Forbes Helen , Handley Pauline S. , Loach Diane M. , Tannock Gerald W. , Jenkinson Howard F. 
Published: December 2019

ABSTRACT

It has been shown previously that inactivation of the
cshA
gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium
Streptococcus gordonii
, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral
Actinomyces
species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 ± 14.5 nm long, which are present on wild-type
S. gordonii
DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of
cshA
mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire
cshA
gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous
Enterococcus faecalis
JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over
E. faecalis
JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in
E. faecalis
also promoted cell-cell aggregation and increased the ability of enterococci to bind
Actinomyces naeslundii
cells. Electron micrographs of negatively stained
E. faecalis
cells expressing CshA showed peritrichous surface fibrils 70.3 ± 9.1 nm long that were absent from control
E. faecalis
JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of
E. faecalis
cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of
S. gordonii
adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral “
sanguis
-like” streptococci.
Molecular Characterization of KatY (Antigen 5), a Thermoregulated Chromosomally Encoded Catalase-Peroxidase ofYersinia pestis
Authors: Garcia Emilio , Nedialkov Yuri A. , Elliott Jeffrey , Motin Vladimir L. , Brubaker Robert R. 
Published: December 2019

ABSTRACTThe first temperature-dependent proteins (expressed at 37°C, but not 26°C) to be identified inYersinia pestiswere antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared byYersinia pseudotuberculosis, but notYersinia enterocolitica. We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes ofY. pestiswas enriched for anti-KatY. A sequenced ∼16-kbY. pestisDNA insert of a positive pLG338 clone indicated thatkatYencodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6.43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG inEscherichia coli) and is closely related to plasmid-encoded KatP of enterohemorrhagicE. coliO157:H7 (75% identity).katYencoded a putative Ca2+-binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF.rbsAwas located upstream ofkatY, andcybB,cybC,dmsABC, andaraDwere mapped downstream. These genes are not linked tokatGorkatPinE. coli.
Peptidoglycan Hydrolase LytF Plays a Role in Cell Separation with CwlF during Vegetative Growth of
Bacillus subtilis
Authors: Ohnishi Ryo , Ishikawa Shu , Sekiguchi Junichi 
Published: December 2019

ABSTRACT

Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the
yhdD
gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of
Listeria monocytogenes
. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in
Escherichia coli
cells hydrolyzed the linkage of
d
-γ-glutamyl-
meso
-diaminopimelic acid in murein peptides, indicating that it is a
d,l
-endopeptidase. Northern hybridization and primer extension analyses indicated that the
lytF
gene was transcribed by Eς
D
RNA polymerase. Disruption of
lytF
led to slightly filamentous cells, and a
lytF cwlF
double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the
cwlF sigD
mutant.
Differential Expression of Nonagglutinating Fimbriae and MR/P Pili in Swarming Colonies of
Proteus mirabilis
Authors: Latta Roger K. , Grondin Annic , Jarrell Harold C. , Nicholls G. Rod , Bérubé Luc R. 
Published: December 2019

ABSTRACT

The expression of nonagglutinating fimbriae (NAF) and mannose-resistant/
Proteus
-like (MR/P) pili in swarming colonies of
Proteus mirabilis
was investigated. Elongated swarmer cells do not express pili, and the relative number of bacteria expressing NAF during swarming and early consolidation phases was very low (<5%). Relative expression of NAF in a terrace increased to approximately 30% at 48 h. We also determined the expression of NAF and MR/P pili in two phenotypically distinguishable regions of each terrace. The expression of both NAF and MR/P pili was always higher in the region closer (proximal) to the middle of the colony than in the distal region of the terrace. The relative numbers of bacteria expressing NAF or MR/P pili in the proximal region were between 39.1 and 63% and between 5.9 and 7.7%, respectively. In the distal region, expression levels were between 20.8 and 27.3% and between 3.7 and 5.6%, respectively. A time course experiment testing NAF expression in both the proximal and distal regions of a terrace indicated that NAF expression in the proximal regions was always higher than in the distal regions and increased to a plateau 40 to 50 h after the start of the swarming phase for any given terrace. These results indicate that expression of NAF or MR/P pili in swarming colonies of
P. mirabilis
is highly organized, spatially and temporally. The significance of this controlled differentiation remains to be uncovered.
Isolation and Characterization of Two Cryptic Plasmids in the Ammonia-Oxidizing Bacterium
Nitrosomonas
sp. Strain ENI-11
Authors: Yamagata Akira , Kato Junichi , Hirota Ryuichi , Kuroda Akio , Ikeda Tsukasa , Takiguchi Noboru , Ohtake Hisao 
Published: December 2019

ABSTRACT

Two plasmids were discovered in the ammonia-oxidizing bacterium
Nitrosomonas
sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a
Bacillus
plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain
Nitrosomonas europaea
IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.
Role of SpoVG in Asymmetric Septation in
Bacillus subtilis
Authors: Matsuno Kiyoshi , Sonenshein Abraham L. 
Published: December 2019

ABSTRACT

Deletion of the
citC
gene, coding for isocitrate dehydrogenase, arrests sporulation of
Bacillus subtilis
at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the
spoVG
gene. The suppressing mutation and other
spoVG
loss-of-function mutations enabled
citC
mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor ς
F
. However, little induction of mother cell-specific, ς
E
-dependent sporulation genes was observed in a
citC spoVG
double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the
citC
mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl
2
. Overexpression of the
spoVG
gene in wild-type cells significantly delayed ς
F
activation. Increased expression and stability of SpoVG in
citC
mutant cells may contribute to the
citC
mutant phenotype. Inactivation of the
spoVG
gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene
minD
, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.
Transcriptional Control of the Iron-Responsive
fxbA
Gene by the Mycobacterial Regulator IdeR
Authors: Dussurget Olivier , Timm Juliano , Gomez Manuel , Gold Benjamin , Yu Shengwei , Sabol Sue Z. , Holmes Randall K. , Jacobs William R. , Smith Issar 
Published: December 2019

ABSTRACT

Exochelin is the primary extracellular siderophore of
Mycobacterium smegmatis
, and the iron-regulated
fxbA
gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557–569, 1994). We investigated the regulation of
fxbA
by the mycobacterial IdeR, a homolog of the
Corynebacterium diphtheriae
iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284–4289, 1995). Gel mobility shift experiments showed that IdeR binds to the
fxbA
regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the
fxbA
promoter that was identified in primer extension assays.
fxbA
regulation was measured in
M. smegmatis
wild-type and
ideR
mutant strains containing
fxbA
promoter-
lacZ
fusions. These experiments confirmed that
fxbA
expression is negatively regulated by iron and showed that inactivation of
ideR
results in iron-independent expression of
fxbA
. However, the levels of its expression in the
ideR
mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of
fxbA
.
Transcriptional Regulation of the
esp
Genes of Enterohemorrhagic
Escherichia coli
Authors: Beltrametti Fabrizio , Kresse Andreas U. , Guzmán Carlos A. 
Published: December 2019

ABSTRACT

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic
Escherichia coli
(EHEC) are organized in a single operon. The
esp
operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the
espA
gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca
2+
, Mn
2+
, and HEPES. Transcription of the
esp
operon seems to be switched off in tightly attached bacteria. The activation process is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influenced by the degree of DNA supercoiling. Transcription is ς
S
dependent, and the H-NS protein contributes to its fine tuning. Identification of the factors involved in activation of the
esp
operon and the signals responsible for modulation may facilitate understanding of the underlying molecular events leading to sequential expression of virulence factors during natural infections caused by EHEC.
Role of Genomic Rearrangements in Producing New Ribotypes of
Salmonella typhi
Authors: Ng Ivy , Liu Shu-Lin , Sanderson Kenneth E. 
Published: December 2019

ABSTRACT

Salmonella typhi
is the only species of
Salmonella
which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of
S. typhi
show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease
Pst
I. In addition, the genome of
S. typhi
has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of
rrn
genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of
S. typhi
which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e.g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between
rrnH
and
rrnG
operons. Different genome type groups normally have different ribotypes. The size and identity of the
Pst
I fragment containing each of the seven different
rrn
operons from
S. typhi
Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in
S. typhi
.
Characterization of Colicin S4 and Its Receptor, OmpW, a Minor Protein of the
Escherichia coli
Outer Membrane
Authors: Pilsl Holger , Smajs David , Braun Volkmar 
Published: December 2019

ABSTRACT

Analysis of the nucleotide sequence of an
Escherichia coli
colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.
A Role for the Mannose-Sensitive Hemagglutinin in Biofilm Formation by
Vibrio cholerae
El Tor
Authors: Watnick Paula I. , Fullner Karla Jean , Kolter Roberto 
Published: December 2019

ABSTRACT

While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen’s survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that
Vibrio cholerae
El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of
V. cholerae
to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces.
A
Bacillus subtilis
Secreted Protein with a Role in Endospore Coat Assembly and Function
Authors: Serrano Mónica , Zilhão Rita , Ricca Ezio , Ozin Amanda J. , Moran Charles P. , Henriques Adriano O. 
Published: December 2019

ABSTRACT

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In
Bacillus subtilis
, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a
gerE
mutant. The N-terminal sequence of this polypeptide matched the deduced product of the
tasA
gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of
tasA
results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that
tasA
and
gerE
mutations act synergistically to decrease the efficiency of spore germination. We show that
tasA
is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the
tasA
operon is enhanced 2 h after the onset of sporulation, under the control of ς
H
. When
tasA
transcription is uncoupled from
sipW
expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.
Characterization of the
vanD
Glycopeptide Resistance Gene Cluster from
Enterococcus faecium
BM4339
Authors: Casadewall Barbara , Courvalin Patrice 
Published: December 2019

ABSTRACT

VanD-type resistance to glycopeptides in
Enterococcus faecium
BM4339 is due to constitutive synthesis of
d
-alanyl-
d
-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016–2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3′ distal part encoded the VanH
D
dehydrogenase, the VanD ligase, and the VanX
D
dd
-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanY
D
protein was homologous to penicillin-binding proteins that display
dd
-carboxypeptidase activity. The 5′ end coded for the putative VanR
D
-VanS
D
two-component regulatory system. Due to a frameshift mutation in the chromosomal
ddl
gene, BM4339 produced an impaired
d
-alanine:
d
-alanine ligase. However, since expression of the resistance genes is constitutive, growth of
E. faecium
BM4339 was not dependent on the presence of glycopeptides in the culture medium.
A Response Regulator That Represses Transcription of Several Virulence Operons in the Group A Streptococcus
Authors: Federle Michael J. , McIver Kevin S. , Scott June R. 
Published: December 2019

ABSTRACTA search for homologs of theBacillus subtilisPhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified ascsrR(J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209–219, 1998), caused the strain to produce mucoid colonies and to increase transcription ofhasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription ofska(encoding streptokinase),sagA(streptolysin S), andspeMF(mitogenic factor) but did not affect transcription ofslo(streptolysin O),mga(multiple gene regulator of GAS),emm(M protein),scpA(complement C5a peptidase), orspeBorspeC(pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system inEscherichia coliwhose mechanism is unrelated to that of these genes in GAS, the locus has been renamedcovR, for “control of virulence genes” in GAS. Transcription of thecovRoperon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of thecovRnonpolar insertion mutant were complemented by thecovRgene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.