bluuebunny/crossref_metadata_2025 huggingface dataset:
Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the
Escherichia coli
Export Chaperone SecB
Authors: Cook Heather A. , Kumamoto Carol A.
Published: December 2019

SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins in
Escherichia coli
. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the
secA
locus that compensate for the export defect caused by the
secB
missense mutation
secBL75Q
were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.
Identification and Transcriptional Control of the Genes Encoding the
Caulobacter crescentus
ClpXP Protease
Authors: Østerås Magne , Stotz Agathe , Nuoffer Stefanie Schmid , Jenal Urs
Published: December 2019

The region of the
Caulobacter crescentus
chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the
C. crescentus
ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The
C. crescentus clpX
gene was shown by complementation analysis to be functional in
Escherichia coli
. However,
clpX
from
E. coli
was not able to substitute for the essential nature of the
clpX
gene in
C. crescentus
. The
clpP
and
clpX
genes are separated on the
C. crescentus
chromosome by an open reading frame pointing in the opposite direction from the
clp
genes, and transcription of
clpP
and
clpX
was found to be uncoupled.
clpP
is transcribed as a monocistronic unit with a promoter (P
P1
) located immediately upstream of the 5′ end of the gene and a terminator structure following its 3′ end. P
P1
is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for
clpX
(P
X1
, P
X2
, and P
X3
) were mapped in the
clpP-clpX
intergenic region. In contrast to P
P1
, the
clpX
promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the
clpP
and
clpX
promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in
C. crescentus
. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.
Role of the Mitogen-Activated Protein Kinase Hog1p in Morphogenesis and Virulence of
Candida albicans
Authors: Alonso-Monge R. , Navarro-García F. , Molero G. , Diez-Orejas R. , Gustin M. , Pla J. , Sánchez M. , Nombela C.
Published: December 2019

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in
Candida albicans
was addressed through the characterization of
C. albicans
strains without a functional
HOG1
gene. Analysis of the phenotype of
hog1
mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions,
hog1
mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the
hog1
mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in
C. albicans
. We show that
C. albicans hog1
mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that
HOG1
overexpression in
Saccharomyces cerevisiae
represses the pseudodimorphic transition. Most interestingly, deletion of
HOG1
resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.
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

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

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

The 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

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

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

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

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

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

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

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

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

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

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

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

A 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.
The ATP-Dependent HslVU/ClpQY Protease Participates in Turnover of Cell Division Inhibitor SulA in
Escherichia coli
Authors: Kanemori Masaaki , Yanagi Hideki , Yura Takashi
Published: December 2019

Escherichia coli
mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [Δ
hslVU
and Δ(
clpPX-lon
)], cannot grow at low (30°C) or very high (45°C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30°C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either
ftsZ
(encoding a key cell division protein) or
sulA
(encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the
ftsZ
mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the
sulA
mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Δ(
clpPX-lon
) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.
Insertional Inactivation of
Treponema denticola tap1
Results in a Nonmotile Mutant with Elongated Flagellar Hooks
Authors: Limberger Ronald J. , Slivienski Linda L. , Izard Jacques , Samsonoff William A.
Published: December 2019

The treponemal
fla
operon is comprised of numerous motility-related genes; however, the initial gene of this operon,
tap1
, has no known function. A recently developed system to generate specific mutants in
Treponema denticola
was utilized to determine if Tap1 was essential for motility.
T. denticola tap1
and flanking DNA were identified, cloned, and sequenced, and a suicide plasmid that contained
tap1
interrupted with an erythromycin resistance cassette (
ermF
and
ermAM
) was constructed. Because of potential polar effects from this cassette, a second plasmid that contained
tap1
interrupted with a modified erythromycin resistance cassette that lacked the putative
ermF
transcription terminator was constructed. Electroporation-mediated allelic exchange incorporated the interrupted
tap1
genes into the
T. denticola
chromosome, creating Tap1-deficient mutants. Reverse transcriptase PCR revealed that the erythromycin resistance cassette within
tap1
did not terminate
fla
operon transcription in either mutant. Moreover, the phenotypes of the two mutants were indistinguishable. These mutants lacked motion in liquid culture, were unable to spread on agar plates, and lacked flagellar filaments as determined by electron microscopy. Immunoblots revealed a marked reduction in detectable FlaB flagellar filament protein compared to that of wild type; however,
flaB
RNA was easily detectable, and transcription levels did not appear to be altered. The basis for the lack of filament protein expression is unknown. Immunoblotting also showed that the flagellar hook protein (FlgE) was synthesized in the Tap1-deficient mutant; however, electron microscopy revealed that the mutant possessed unusual elongated hooks of variable lengths. We propose that treponemal Tap1 is analogous to FliK, which is involved in monitoring the flagellar hook length of
Salmonella typhimurium.
A Role for a Highly Conserved Protein of Unknown Function in Regulation of
Bacillus subtilis purA
by the Purine Repressor
Authors: Rappu Pekka , Shin Byung Sik , Zalkin Howard , Mäntsälä Pekka
Published: December 2019

Regulation of the purine biosynthetic gene
purA
was examined by using a transcriptional fusion to a luciferase reporter gene. Transcription was repressed about 10-fold by the addition of adenine and increased approximately 4.5-fold by the addition of guanosine. This regulation is mediated by a purine repressor (PurR). In a
purR
mutant, basal expression was increased 10-fold, and there was no further stimulation by guanosine or repression by adenine. An open reading frame,
yabJ
, immediately downstream from
purR
was found to have a role in the repression of
purA
by adenine. Repression by adenine was perturbed in a

purR
+
yabJ

mutant, although guanosine regulation was retained. Mutations in the PurR PRPP binding motif abolished guanosine regulation in the
yabJ
mutant. Thus, PRPP appears to be required for upregulation by guanosine. The amino acid sequence of YabJ is homologous to the YER057c/YjgF protein family of unknown function.
Cloning and Characterization of the Gene EncodingPasteurella haemolyticaFnrP, a Regulator of theEscherichia coliSilent Hemolysin SheA
Authors: Uhlich Gaylen A. , McNamara Peter J. , Iandolo John J. , Mosier Derek A.
Published: December 2019

APasteurella haemolyticaA1 gene was identified from a recombinant library clone that expressed hemolysis in hostEscherichia colicells. The gene, designatedfnrP, had sequence identity toE. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of afnr-null mutant strain ofE. coliand increased expression of the Fnr-dependent, anaerobic terminal reductase gene,frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed inE. coli ΔsheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.
Uptake of Pyocin S3 Occurs through the Outer Membrane Ferripyoverdine Type II Receptor of
Pseudomonas aeruginosa
Authors: Baysse Christine , Meyer Jean-Marie , Plesiat Patrick , Geoffroy Valérie , Michel-Briand Yvon , Cornelis Pierre
Published: December 2019

Pyocin S3 was found to kill exclusively
Pseudomonas aeruginosa
isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn
5
mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.
Effect of
rpoS
Mutation on the Stress Response and Expression of Virulence Factors in
Pseudomonas aeruginosa
Authors: Suh Sang-Jin , Silo-Suh Laura , Woods Donald E. , Hassett Daniel J. , West Susan E. H. , Ohman Dennis E.
Published: December 2019

The sigma factor RpoS (ς
S
) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in
Pseudomonas aeruginosa
physiology and pathogenesis, we constructed
rpoS
mutants in several strains of
P. aeruginosa
, including PAO1. The PAO1
rpoS
mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1
rpoS
mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1
rpoS
mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H
2
O
2
. In accordance with the hypersensitivity to H
2
O
2
, catalase production was 60% lower in the
rpoS
mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of
P. aeruginosa
. The
rpoS
mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in
rpoS
in PAO1. The
rpoS
mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1
rpoS
mutant that was observed in a rat chronic-lung-infection model. In addition, the
rpoS
mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the
rpoS101
::
aacCI
mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1
rpoS
mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in
P. aeruginosa
physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.
Expression of the ς
B
-Dependent General Stress Regulon Confers Multiple Stress Resistance in
Bacillus subtilis
Authors: Völker Uwe , Maul Björn , Hecker Michael
Published: December 2019

The alternative sigma factor ς
B
of
Bacillus subtilis
is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in
sigB
on the stress and starvation survival of
B. subtilis. sigB
mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54°C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with ς
B
-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the ς
B
response, but surprisingly, ς
B
did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of ς
B
-dependent genes provides nonsporulated
B. subtilis
cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.
Interaction of
Azospirillum lipoferum
with Wheat Germ Agglutinin Stimulates Nitrogen Fixation
Authors: Karpati Eva , Kiss Peter , Ponyi Tamas , Fendrik Istvan , de Zamaroczy Miklos , Orosz Laszlo
Published: December 2019

In vitro, the nitrogen fixation capability of
A. lipoferum
is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect required
N
-acetyl-
d
-glucosamine dimer (GlcNAc
di
) terminated sugar side chains of the receptor and was dependent on the number of GlcNAc
di
links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the
nifH
and
nifA
genes and of the
glnBA
gene cluster but not of the
glnA
gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.
Analysis of Peptidoglycan Structure from Vegetative Cells of
Bacillus subtilis
168 and Role of PBP 5 in Peptidoglycan Maturation
Authors: Atrih Abdelmadjid , Bacher Gerold , Allmaier Günter , Williamson Michael P. , Foster Simon J.
Published: December 2019

The composition and fine structure of the vegetative cell wall peptidoglycan from
Bacillus subtilis
were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in
B. subtilis
vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of
B. subtilis
muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total.
B. subtilis
peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a
dacA
(PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the
dacA
mutant. This major low-molecular-weight PBP (
dd
-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.
Escherichia coli
Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis
Authors: Denome Sylvia A. , Elf Pamela K. , Henderson Thomas A. , Nelson David E. , Young Kevin D.
Published: December 2019

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of
Escherichia coli
, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two
res
sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of
E. coli
via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in
trans
, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp
res
site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the
dacD
gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
Characterization of the
Moraxella catarrhalis uspA1
and
uspA2
Genes and Their Encoded Products
Authors: Cope Leslie D. , Lafontaine Eric R. , Slaughter Clive A. , Hasemann Charles A. , Aebi Christoph , Henderson Frederick W. , McCracken George H. , Hansen Eric J.
Published: December 2019

The
uspA1
and
uspA2
genes of
M. catarrhalis
O35E encode two different surface-exposed proteins which were previously shown to share a 140-amino-acid region with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367–4377, 1997). The N-terminal amino acid sequences of the mature forms of both UspA1 and UspA2 from strain O35E were determined after enzymatic treatment to remove the N-terminal pyroglutamyl residue that had blocked Edman degradation. Mass spectrometric analysis indicated that the molecular mass of UspA1 from
M. catarrhalis
O35E was 83,500 ± 116 Da. Nucleotide sequence analysis of the
uspA1
and
uspA2
genes from three other
M. catarrhalis
strains (TTA24, ATCC 25238, and V1171) revealed that the encoded protein products were very similar to those from strain O35E. Western blot analysis was used to confirm that each of these three strains of
M. catarrhalis
expressed both UspA1 and UspA2 proteins. Several different and repetitive amino acid motifs were present in both UspA1 and UspA2 from these four strains, and some of these were predicted to form coiled coils. Linear DNA templates were used in an in vitro transcription-translation system to determine the sizes of the monomeric forms of the UspA1 and UspA2 proteins from strains O35E and TTA24.