bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTAPasteurella 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.
Nonribosomal Peptide Synthesis and Toxigenicity of Cyanobacteria
Authors: Neilan Brett A. , Dittmann Elke , Rouhiainen Leo , Bass R. Amanda , Schaub Verena , Sivonen Kaarina , Börner Thomas 
Published: December 2019

ABSTRACT
Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546–1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69–72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.
Identification of a Putative P-Transporter Operon in the Genome of aBurkholderiaStrain Living inside the Arbuscular Mycorrhizal FungusGigaspora margarita
Authors: Ruiz-Lozano J. M. , Bonfante P. 
Published: December 2019

ABSTRACTThis article reports the identification of a putative P-transporter operon in the genome of aBurkholderiasp. living in the cytoplasm of the arbuscular mycorrhizal fungusGigaspora margarita. Its presence suggests thatBurkholderiasp. has the potential for P uptake from this environment. This finding raises new questions concerning the importance of intracellular bacteria for mycorrhizal symbiosis.
The
pvc
Gene Cluster of
Pseudomonas aeruginosa
: Role in Synthesis of the Pyoverdine Chromophore and Regulation by PtxR and PvdS
Authors: Stintzi Alain , Johnson Zaiga , Stonehouse Martin , Ochsner Urs , Meyer Jean-Marie , Vasil Michael L. , Poole Keith 
Published: December 2019

ABSTRACT

A putative operon of four genes implicated in the synthesis of the chromophore moiety of the
Pseudomonas aeruginosa
siderophore pyoverdine, dubbed
pvcABCD
(where
pvc
stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the
pvc
genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore.
pvcABCD
expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.
First Chromosomal Restriction Map ofActinobacillus pleuropneumoniaeand Localization of Putative Virulence-Associated Genes
Authors: Oswald Winfried , Konine Denis V. , Rohde Judith , Gerlach Gerald-F. 
Published: December 2019

ABSTRACTCombined physical and genetic maps of the genomes ofActinobacillus pleuropneumoniaeAP76 (serotype 7 clinical isolate) and ofA. pleuropneumoniaeATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleasesApaI,AscI,NotI, andSalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between theApaI,AscI,NotI, andSalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The singleSalI site within the chromosome of strainA. pleuropneumoniaeAP76 was defined as position 1 of the map; for the map ofA. pleuropneumoniaeATCC 27088, the correspondingSalI site was chosen. Putative virulence-associated genes (apx,omlA,sodA,tbpBA,ureC, and a repeat element) and housekeeping genes (glyA,metJ,recA, andrhoAP) were positioned on the physical maps and located on theApaI andNotI fragments ofA. pleuropneumoniaeserotype reference strains.