bluuebunny/crossref_metadata_2025_split huggingface dataset:
CS5 Pilus Biosynthesis Genes from Enterotoxigenic
Escherichia coli
O115:H40
Authors: Duthy Thomas G. , Staendner Lothar H. , Manning Paul A. , Heuzenroeder Michael W. 
Published: December 2019

ABSTRACT

We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic
Escherichia coli
O115:H40 downstream of the major subunit gene, designated
csfA
(for
coli surface factor five
A). Five more open reading frames (ORFs) (
csfB
,
csfC
,
csfE
,
csfF
, and
csfD
) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned
csf
ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the
csf
region in
E. coli
K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.
An Archaeal Aminoacyl-tRNA Synthetase Missing from Genomic Analysis
Authors: Hamann Christian S. , Sowers Kevin R. , Lipman Richard S. A. , Hou Ya-Ming 
Published: December 2019

ABSTRACT

The complete genomic sequencing of
Methanococcus jannaschii
cannot identify the gene for the cysteine-specific member of aminoacyl-tRNA synthetases. However, we show here that enzyme activity is present in the cell lysate of
M. jannaschii
. The demonstration of this activity suggests a direct pathway for the synthesis of cysteinyl-tRNA
Cys
during protein synthesis.
Regulation of Transfer Functions by the
imp
Locus of the
Streptomyces coelicolor
Plasmidogenic Element SLP1
Authors: Hagege Juliette M. , Brasch Michael A. , Cohen Stanley N. 
Published: December 2019

ABSTRACT

SLP1

int

is a 17.2-kb genetic element that normally is integrated site specifically into the chromosome of
Streptomyces coelicolor
A3(2). The
imp
operon within SLP1

int

represses replication of both chromosomally integrated and extrachromosomal SLP1. During mating with
S. lividans
, SLP1

int

can excise, delete part of
imp
, and form a family of autonomously replicating conjugative plasmids. Earlier work has shown that
impA
and
impC
gene products act in concert to control plasmid maintenance and regulate their own transcription. Here we report that these
imp
genes act also on a second promoter, P

op
imp

(promoter opposite
imp
), located adjacent to, and initiating transcription divergent from,
imp
to regulate loci involved in the intramycelial transfer of SLP1 plasmids.
spdB1
and
spdB2
, two overlapping genes immediately 3′ to P

op
imp

and directly regulated by
imp
, are shown by Tn
5
mutagenesis to control transfer-associated growth inhibition (i.e., pocking). Additional genes resembling transfer genes of other
Streptomyces
spp. plasmids and required for SLP1 transfer and/or postconjugal intramycelial spread are located more distally to P

op
imp

. Expression of
impA
and
impC
in an otherwise competent recipient strain prevented SLP1-mediated gene transfer of chromosomal and plasmid genes but not plasmid-independent chromosome-mobilizing activity, suggesting that information transduced to recipients after the formation of mating pairs affects
imp
activity. Taken together with earlier evidence that the
imp
operon regulates SLP1 DNA replication, the results reported here implicate
imp
in the overall regulation of functions related to the extrachromosomal state of SLP1.
Identification of
pel
, a
Streptococcus pyogenes
Locus That Affects both Surface and Secreted Proteins
Authors: Li Zhuqing , Sledjeski Darren D. , Kreikemeyer Bernd , Podbielski Andreas , Boyle Michael D. P. 
Published: December 2019

ABSTRACT

A Tn
917
insertion mutant of an M49 serotype, opacity factor-positive
Streptococcus pyogenes
, was isolated. It had the following phenotypes: decreased β-hemolysis mediated by streptolysin S, reduction in the activity of a secreted cysteine protease and streptokinase, and an altered immunoglobulin and fibrinogen-binding phenotype. The site of insertion of Tn
917
into the chromosome and the surrounding sequence, the
pel
region (pleiotropic effect locus), was determined. Phage A25 transduction confirmed that the pleiotropic changes in phenotype could be cotransduced with Tn
917
. The
pel
region was cloned and sequenced, and the transposon was found to be inserted upstream of a single open reading frame which led to a failure to transcribe a 500-base mRNA. The loss of this transcript decreased the transcription of
emm
and
speB
genes and reduced the secretion of streptokinase. Enhanced Pel expression from a nisin-inducible plasmid resulted in increased message levels for
emm
in a wild-type organism. Characterization of the
pel
mutant provides evidence for the coordinated regulation of secreted and surface proteins and suggests the existence of a new global regulatory factor in
S. pyogenes
.
Phylogenetic Analysis of L4-Mediated Autogenous Control of the S10 Ribosomal Protein Operon
Authors: Allen Todd , Shen Ping , Samsel Leigh , Liu Raymond , Lindahl Lasse , Zengel Janice M. 
Published: December 2019

ABSTRACT

We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes
Escherichia coli
. In
E. coli
, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (
Salmonella typhimurium
,
Citrobacter freundii
,
Yersinia enterocolitica
,
Serratia marcescens
, and
Morganella morganii
) and two nonenteric members of the gamma subdivision (
Haemophilus influenzae
and
Vibrio cholerae
) shows that these foreign leaders share significant structural homology with the
E. coli
leader, particularly in the region which is critical for L4-mediated autogenous control in
E. coli
. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in
E. coli
. Our results suggest that an
E. coli
-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one,
Pseudomonas aeruginosa
, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in
E. coli
. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.
Analysis of the
Corynebacterium glutamicum dapA
Promoter
Authors: Vašicová Pavla , Pátek Miroslav , Nešvera Jan , Sahm Hermann , Eikmanns Bernhard 
Published: December 2019

ABSTRACT

Deletion and mutational analysis of the promoter P-
dapA
from
Corynebacterium glutamicum
was performed to identify regions and particular nucleotides important for its function. An extended −10 region and a stretch of six T’s at positions −55 to −50 were found to be the most important elements in the promoter function. The results of mutational analysis of P-
dapA
are consistent with the conclusions of statistical computer-aided analysis of 44
C. glutamicum
promoter sequences.
Differential Stabilities of Phosphorylated Response Regulator Domains Reflect Functional Roles of the Yeast Osmoregulatory SLN1 and SSK1 Proteins
Authors: Janiak-Spens Fabiola , Sparling Jeffrey M. , Gurfinkel Michael , West Ann H. 
Published: December 2019

ABSTRACT

Osmoregulation in
Saccharomyces cerevisiae
involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in
Bacillus subtilis
and anaerobic respiration in
Escherichia coli
. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from
E. coli
. A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.
Helicobacter pylori
: a Eubacterium Lacking the Stringent Response
Authors: Scoarughi Gian Luca , Cimmino Carmen , Donini Pierluigi 
Published: December 2019

ABSTRACT

Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of
Helicobacter pylori
. All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA. This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype. The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms. However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism. These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches.
Effect of ppGpp on
Escherichia coli
Cyclopropane Fatty Acid Synthesis Is Mediated through the RpoS Sigma Factor (ς
S
)
Authors: Eichel Johannes , Chang Ying-Ying , Riesenberg Dieter , Cronan John E. 
Published: December 2019

ABSTRACT

Strains of
Escherichia coli
carrying mutations at the
relA
locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3′,5′-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification. We report that the effect of RelA on CFA synthesis is indirect. In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity. The
relA
effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase.
Organized Cell Swimming Motions in
Bacillus subtilis
Colonies: Patterns of Short-Lived Whirls and Jets
Authors: Mendelson Neil H. , Bourque Adrienne , Wilkening Kathryn , Anderson Kevin R. , Watkins Joseph C. 
Published: December 2019

ABSTRACT

The swimming motions of cells within
Bacillus subtilis
colonies, as well as the associated fluid flows, were analyzed from video films produced during colony growth and expansion on wet agar surfaces. Individual cells in very wet dense populations moved at rates between 76 and 116 μm/s. Swimming cells were organized into patterns of whirls, each approximately 1,000 μm
2
, and jets of about 95 by 12 μm. Whirls and jets were short-lived, lasting only about 0.25 s. Patterns within given areas constantly repeated with a periodicity of approximately 1 s. Whirls of a given direction became disorganized and then re-formed, usually into whirls moving in the opposite direction. Pattern elements were also organized with respect to one another in the colony. Neighboring whirls usually turned in opposite directions. This correlation decreased as a function of distance between whirls. Fluid flows associated with whirls and jets were measured by observing the movement of marker latex spheres added to colonies. The average velocity of markers traveling in whirls was 19 μm/s, whereas those traveling in jets moved at 27 μm/s. The paths followed by markers were aligned with the direction of cell motion, suggesting that cells create flows moving with them into whirls and along jets. When colonies became dry, swimming motions ceased except in regions close to the periphery and in isolated islands where cells traveled in slow whirls at about 4 μm/s. The addition of water resulted in immediate though transient rapid swimming (> 80 μm/s) in characteristic whirl and jet patterns. The rate of swimming decreased to 13 μm/s within 2 min, however, as the water diffused into the agar. Organized swimming patterns were nevertheless preserved throughout this period. These findings show that cell swimming in colonies is highly organized.
Cloning and Characterization of a Tetracycline Resistance Determinant Present in
Agrobacterium tumefaciens
C58
Authors: Luo Zhao-Qing , Farrand Stephen K. 
Published: December 2019

ABSTRACT

Agrobacterium tumefaciens
C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in
Escherichia coli
and
Agrobacterium
was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes,
tetA
and
tetR
, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant,
tetR
was disrupted by an IS
426
. The homologous region from wild-type NT1 contained an intact
tetR
gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus
Agrobacterium
surveyed, only strains C58 and T37 contained the
tet
determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this
tet
gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this
tet
gene system. The divergent promoter region of this
tet
system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the
tet
determinant from the IncP1α R plasmid RP4. TetR repressor proteins from the
Agrobacterium tet
system and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetR
C58
) on the
tetA
gene from strain RP4 (
tetA
RP4
) was not relieved by tetracycline, repression of
tetA
C58
by TetR
RP4
was lifted by this antibiotic.
A Plant-Type (β-Class) Carbonic Anhydrase in the Thermophilic MethanoarchaeonMethanobacterium thermoautotrophicum
Authors: Smith Kerry S. , Ferry James G. 
Published: December 2019

ABSTRACTCarbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (β-class) carbonic anhydrase in the archaea. TheMethanobacterium thermoautotrophicumΔHcabgene was hyperexpressed inEscherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75°C. No esterase activity was detected withp-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO2hydration activity with akcatof 1.7 × 104s−1andKmfor CO2of 2.9 mM at pH 8.5 and 25°C. Western blot analysis indicates that Cab (β class) is expressed inM. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the γ carbonic anhydrase fromMethanosarcina thermophilawas detected. These results show that β-class carbonic anhydrases extend not only into theArchaeadomain but also into the thermophilic prokaryotes.
Characterization of MexT, the Regulator of the MexE-MexF-OprN Multidrug Efflux System of
Pseudomonas aeruginosa
Authors: Köhler Thilo , Epp Simone F. , Curty Lasta Kocjancic , Pechère Jean-Claude 
Published: December 2019

ABSTRACT

We investigated the regulation of the MexEF-OprN multidrug efflux system of
Pseudomonas aeruginosa
, which is overexpressed in
nfxC
-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the
mexEF-oprN
operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in
Escherichia coli
from a plasmid carrying the
mexT
gene. Expression of a
mexE
::
lacZ
fusion was 10-fold higher in
nfxC
-type mutants than in the wild-type strain; however, transcription of
mexT
as well as the
mexT
DNA region was unchanged. Located adjacent to
mexT
but transcribed in opposite direction, the beginning of an ORF termed
qrh
(quinone oxidoreductase homologue) was identified. Expression of a
qrh
::
lacZ
fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of
nfxC
-type mutants to carbapenem antibiotics.
In Vitro Characterization of Peptidoglycan-Associated Lipoprotein (PAL)–Peptidoglycan and PAL–TolB Interactions
Authors: Bouveret Emmanuelle , Bénédetti Hélène , Rigal Alain , Loret Erwann , Lazdunski Claude 
Published: December 2019

ABSTRACT

The Tol-peptidoglycan-associated lipoprotein (PAL) system of
Escherichia coli
is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.
Hybrid Motor with H
+
- and Na
+
-Driven Components Can Rotate
Vibrio
Polar Flagella by Using Sodium Ions
Authors: Asai Yukako , Kawagishi Ikuro , Sockett R. Elizabeth , Homma Michio 
Published: December 2019

ABSTRACT

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium
Vibrio alginolyticus
is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium
Rhodobacter sphaeroides
. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA.
R. sphaeroides
MotB has high similarity only to the transmembrane region of PomB. To examine whether the
R. sphaeroides
motor genes can function in place of the
pomA
and
pomB
genes of
V. alginolyticus
, we constructed plasmids including both
motA
and
motB
or
motA
alone and transformed them into missense and null
pomA
-paralyzed mutants of
V. alginolyticus
. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand,
pomB
mutants were not restored to motility by any plasmid containing
motA
and/or
motB
. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of
R. sphaeroides
can generate torque by coupling with the sodium ion flux in place of PomA of
V. alginolyticus.
Pathogenic
Yersinia
Species Carry a Novel, Cold-Inducible Major Cold Shock Protein Tandem Gene Duplication Producing both Bicistronic and Monocistronic mRNA
Authors: Neuhaus Klaus , Francis Kevin P. , Rapposch Sonja , Görg Angelika , Scherer Siegfried 
Published: December 2019

ABSTRACT

Inverse PCR was used to amplify major cold shock protein (MCSP) gene families from a diverse range of bacteria, including the psychrotolerant
Yersinia enterocolitica
, which was found to have two almost identical MCSP coding regions (
cspA1
and
cspA2
) located approximately 300 bp apart. This tandem gene duplication was also found in
Y. pestis
,
Y. pseudotuberculosis
, and
Y. ruckeri
but not in other bacteria. Analysis of the transcriptional regulation of this MCSP gene in
Y. enterocolitica
, performed by using both reverse transcriptase-PCR and Northern blot assays, showed there to be two cold-inducible mRNA templates arising from this locus: a monocistronic template of approximately 450 bp (
cspA1
) and a bicistronic template of approximately 900 bp (
cspA1/A2
). The former may be due to a secondary structure between
cspA1
and
cspA2
causing either 3′ degradation protection of
cspA1
or, more probably, partial termination after
cspA1
. Primer extension experiments identified a putative transcriptional start site (+1) which is flanked by a cold-box motif and promoter elements (−10 and −35) similar to those found in
Escherichia coli
cold-inducible MCSP genes. At 30°C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10°C, transcription of the bicistronic mRNA was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was apparent after only 10 min of cold shock from 30°C to 10°C. The data demonstrate an extraordinary capacity of the psychrotolerant
Y. enterocolitica
to produce major cold shock proteins upon cold shock.