bluuebunny/crossref_metadata_2025 huggingface dataset:
Elements Involved in Catabolite Repression and Substrate Induction of the Lactose Operon in
Lactobacillus casei
Authors: Gosalbes María José , Monedero Vicente , Pérez-Martínez Gaspar
Published: December 2019

In
Lactobacillus casei
ATCC 393, the chromosomally encoded lactose operon,
lacTEGF
, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-β-galactosidase.
lacT
,
lacE
, and
lacF
mutant strains were constructed by double crossover. The
lacT
strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas
lacE
and
lacF
mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of
lac
promoter (
lacp
) and
lacpΔRAT
with the
Escherichia coli gusA
gene in the different
lac
mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a
ccpA
mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression of
lacTEGF
was found only in a
man ccpA
double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.
The Extracellular Xylan Degradative System in
Clostridium cellulolyticum
Cultivated on Xylan: Evidence for Cell-Free Cellulosome Production
Authors: Mohand-Oussaid O. , Payot S. , Guedon E. , Gelhaye E. , Youyou A. , Petitdemange H.
Published: December 2019

In this study, we demonstrate that the cellulosome of
Clostridium cellulolyticum
grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate,
C. cellulolyticum
is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.
Identification of Site-Specific Recombination Genes
int
and
xis
of the
Rhizobium
Temperate Phage
16-3
Authors: Semsey Szabolcs , Papp IstvAn , Buzas Zsuzsanna , Patthy Andras , Orosz Laszlo , Papp Peter P.
Published: December 2019

Phage
16-3
is a temperate phage of
Rhizobium meliloti
41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of
attB
and
attP
. Here we report the identification of two phage-encoded genes required for recombinations at these sites:
int
(phage integration) and
xis
(prophage excision). We concluded that Int protein of phage
16-3
belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the
16-3 int xis att
system is not active in
Escherichia coli
, probably due to requirements for host factors that differ in
Rhizobium meliloti
and
E. coli
. The application of the
16-3
site-specific recombination system in biotechnology is discussed.
Identification and Characterization of a New Lipoprotein, NlpI, inEscherichia coliK-12
Authors: Ohara Masaru , Wu Henry C. , Sankaran Krishnan , Rick Paul D.
Published: December 2019

We report here the identification of a new lipoprotein, NlpI, inEscherichia coliK-12. The NlpI structural gene (nlpI) is located between the genespnp(polynucleotide phosphorylase) anddeaD(RNA helicase) at 71 min on theE. colichromosome. ThenlpIgene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. AnnlpI::cmmutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation innlpI, since it was complemented by the wild-typenlpIgene alone. Overexpression of the unalterednlpIgene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.
Regulated Expression of a Highly Conserved Regulatory Gene Cluster Is Necessary for Controlling Photosynthesis Gene Expression in Response to Anaerobiosis in
Rhodobacter capsulatus
Authors: Du Shouying , Kouadio Jean-Louis K. , Bauer Carl E.
Published: December 2019

We utilized primer extension analysis to demonstrate that the divergently transcribed
regB
and
senC-regA-hvrA
transcripts contain stable 5′ ends 43 nucleotides apart within the
regB-senC
intergenic region. DNA sequence analysis indicates that this region contains two divergent promoters with overlapping ς
70
type −35 and −10 promoter recognition sequences. In vivo analysis of expression patterns of
regB
::
lacZ
and
senC-regA-hvrA
::
lacZ
reporter gene fusions demonstrates that the
regB
and
senC-regA-hvrA
transcripts are both negatively regulated by the phosphorylated form of the global response regulator RegA. DNase I protection assays with a constitutively active variant of RegA indicate that RegA binds between
regB
and
senC
overlapping −10 and −35 promoter recognition sequences. Two mutations were also isolated in a
regB
-deficient background that increased expression of the
senC-regA-hvrA
operon 10- and 5-fold, respectively. As a consequence of increased RegA expression, these mutants exhibited elevated aerobic and anaerobic photosynthesis (
puf
) gene expression, even in the absence of the sensor kinase RegB. These results indicate that autoregulation by RegA is a factor contributing to the maintenance of an optimal low level of RegA expression that allows responsiveness to activation by phosphorylation.
Transcriptional Activation of
Agrobacterium tumefaciens
Virulence Gene Promoters in
Escherichia coli
Requires the
A. tumefaciens rpoA
Gene, Encoding the Alpha Subunit of RNA Polymerase
Authors: Lohrke S. M. , Nechaev S. , Yang H. , Severinov K. , Jin S. J.
Published: December 2019

The two-component regulatory system, composed of
virA
and
virG
, is indispensable for transcription of virulence genes within
Agrobacterium tumefaciens
. However,
virA
and
virG
are insufficient to activate transcription from virulence gene promoters within
Escherichia coli
cells, indicating a requirement for additional
A. tumefaciens
genes. In a search for these additional genes, we have identified the
rpoA
gene, encoding the α subunit of RNA polymerase (RNAP), which confers significant expression of a
virB
promoter (
virBp
)::
lacZ
fusion in
E. coli
in the presence of an active transcriptional regulator
virG
gene. We conducted in vitro transcription assays using either reconstituted
E. coli
RNAP or hybrid RNAP in which the α subunit was derived from
A. tumefaciens
. The two forms of RNAP were equally efficient in transcription from a ς
70
-dependent
E. coli gal
P1 promoter; however, only the hybrid RNAP was able to transcribe
virBp
in a
virG
-dependent manner. In addition, we provide evidence that the α subunit from
A. tumefaciens
, but not from
E. coli
, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the α subunit of
A. tumefaciens
and that the α subunit from
E. coli
is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine
vir
gene expression as well as the T-DNA transfer process in
E. coli.
Mutational Analysis and Membrane Topology of ComP, a Quorum-Sensing Histidine Kinase of
Bacillus subtilis
Controlling Competence Development
Authors: Piazza Flavia , Tortosa Pablo , Dubnau David
Published: December 2019

ComP is a sensor histidine kinase of
Bacillus subtilis
required for the signal transduction pathway that initiates the development of competence for genetic transformation. It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the
srfA
promoter. In the present study, fusions to the
Escherichia coli
proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP. These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain. Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction. The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX. The implications of these data are discussed.
The
Bacillus subtilis yaaH
Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores
Authors: Kodama Takeko , Takamatsu Hiromu , Asai Kei , Kobayashi Kazuo , Ogasawara Naotake , Watabe Kazuhito
Published: December 2019

The expression of 21 novel genes located in the region from
dnaA
to
abrB
of the
Bacillus subtilis
chromosome was analyzed. One of the genes,
yaaH
, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that
yaaH
mRNA was first detected from 2 h after the cessation of logarithmic growth (
T
2
) of sporulation in wild-type cells and in
spoIIIG
(SigG
−
) and
spoIVCB
(SigK
−
) mutants but not in
spoIIAC
(SigF
−
) and
spoIIGAB
(SigE
−
) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the
yaaH
gene was produced from
T
2
of sporulation in a
B. subtilis
transformant and extracted from mature spores, indicating that the
yaaH
gene product is a spore protein. Inactivation of the
yaaH
gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of
yaaH
mutant spores in a mixture of
l
-asparagine,
d
-glucose,
d
-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in
l
-alanine-stimulated germination. These results suggest that
yaaH
is a novel gene encoding a spore protein produced in the mother cell compartment from
T
2
of sporulation and that it is required for the
l
-alanine-stimulated germination pathway.
Immunocytochemical Localization of a Calmodulinlike Protein in
Bacillus subtilis
Cells
Authors: Dominguez Delfina C. , Adams Hank , Hageman James H.
Published: December 2019

To determine possible functions of the calmodulinlike protein of
Bacillus subtilis
, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [
45
Ca]CaCl
2
, ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from
B. subtilis
cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles of
B. subtilis
also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.
Alteration of the Repressor Activity of MarR, the Negative Regulator of the
Escherichia coli marRAB
Locus, by Multiple Chemicals In Vitro
Authors: Alekshun Michael N. , Levy Stuart B.
Published: December 2019

MarR negatively regulates expression of the multiple antibiotic resistance operon (
marRAB
) in
Escherichia coli
. In this study, it was demonstrated that sodium salicylate, plumbagin, 2,4-dinitrophenol, and menadione–inducers of the
marRAB
operon in whole cells–all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.
Growth and Glucose Repression Are Controlled by Glucose Transport in
Saccharomyces cerevisiae
Cells Containing Only One Glucose Transporter
Authors: Ye Ling , Kruckeberg Arthur L. , Berden Jan A. , van Dam Karel
Published: December 2019

A set of
Saccharomyces cerevisiae
strains with variable expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the
HXT7
promoter in vitro and integration of the gene at various copy numbers into the genome of an
hxt1-7 gal2
deletion strain. The glucose transport capacity increased in strains with higher levels of
HXT7
expression. The consequences for various physiological properties of varying the glucose transport capacity were examined. The control coefficient of glucose transport with respect to growth rate was 0.54. At high extracellular glucose concentrations, both invertase activity and the rate of oxidative glucose metabolism increased manyfold with decreasing glucose transport capacity, which is indicative of release from glucose repression. These results suggest that the intracellular glucose concentration produces the signal for glucose repression.
Succinate Dehydrogenase (Sdh) from
Bradyrhizobium japonicum
Is Closely Related to Mitochondrial Sdh
Authors: Westenberg David J. , Guerinot Mary Lou
Published: December 2019

The
sdhCDAB
operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont
Bradyrhizobium japonicum
. Sdh from
B. japonicum
is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in
Escherichia coli
.
The
Streptomyces peucetius dpsC
Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide
Authors: Bao Wuli , Sheldon Paul J. , Wendt-Pienkowski Evelyn , Hutchinson C. Richard
Published: December 2019

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of
Streptomyces peucetius
, directly downstream of the genes encoding the β-ketoacyl:acyl carrier protein synthase subunits, are two genes,
dpsC
and
dpsD
, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying
dpsC
and
dpsD
, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by
dpsC
and
dpsD
play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.
Glucose Metabolism in
gcr
Mutants of
Saccharomyces cerevisiae
Authors: Uemura H. , Fraenkel D. G.
Published: December 2019

A
gcr2
null mutant of
Saccharomyces cerevisiae
grows well on glucose in spite of its lower level of glycolytic enzymes between triose phosphates and pyruvate. A quantitative analysis shows that these levels are adequate to the flux but glycerate phosphates are elevated.
Staphylococcus aureus cap5P Encodes a UDP- N -Acetylglucosamine 2-Epimerase with Functional Redundancy
Authors: Kiser Kevin B. , Bhasin Navneet , Deng Lingyi , Lee Jean C.
Published: December 2019

The serotype 5 capsule gene cluster ofStaphylococcus aureuscomprises 16 genes (cap5Athroughcap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that theN-acetylmannosaminuronic acid (ManNAcA) component of theS. aureusserotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded byS. aureus cap5P. Purified Cap5P converted ∼10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression inS. aureuswas unaffected by insertional inactivation ofcap5P. Sequence homology searches of the publicS. aureusgenomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on theS. aureuschromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function inS. aureuswas demonstrated by cloning thecap5Phomologue from strain Newman and complementing anEscherichia coli rffEmutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of theS. aureus cap5Pgene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.
Complementary and Overlapping Selectivity of the Two-Peptide Bacteriocins Plantaricin EF and JK
Authors: Moll Gert N. , van den Akker Emile , Hauge Håvard H. , Nissen-Meyer Jon , Nes Ingolf F. , Konings Wil N. , Driessen Arnold J. M.
Published: December 2019

Plantaricin EF and JK are both two-peptide bacteriocins produced byLactobacillus plantarumC11. The mechanism of plantaricin EF and JK action was studied onL. plantarum965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Δψ) and pH gradient (ΔpH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate thatL. plantarumC11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.
The Adherence-Associated Lipoprotein P100, Encoded by an opp Operon Structure, Functions as the Oligopeptide-Binding Domain OppA of a Putative Oligopeptide Transport System in Mycoplasma hominis
Authors: Henrich Birgit , Hopfe Miriam , Kitzerow Annette , Hadding Ulrich
Published: December 2019

Mycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10.4 kb inM. hominisFBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene andoppBCDFin a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system inM. hominis.
Transcript Cleavage, Attenuation, and an Internal Promoter in the Rhodobacter capsulatus puc Operon
Authors: LeBlanc H. , Lang A. S. , Beatty J. T.
Published: December 2019

The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in thepuf operon. Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII. We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies. Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur. A putative RNA stem-loop structure appears to attenuate transcription initiated at the pucoperon major promoter. We also found that a minorpucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa γ (PucE) protein.
Genome Scanning in Haemophilus influenzae for Identification of Essential Genes
Authors: Reich Karl A. , Chovan Linda , Hessler Paul
Published: December 2019

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames ofHaemophilus influenzaeby PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.
Characterization of the yrbA Gene ofBacillus subtilis, Involved in Resistance and Germination of Spores
Authors: Takamatsu Hiromu , Kodama Takeko , Nakayama Tatsuo , Watabe Kazuhito
Published: December 2019

Insertional inactivation of theyrbAgene ofBacillus subtilisreduced the resistance of the mutant spores to lysozyme. TheyrbAmutant spores lost their optical density at the same rate as the wild-type spores upon incubation withl-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this mediumyrbAspores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis ofyrbAtranscripts in varioussigmutants indicated thatyrbAwas transcribed by RNA polymerase with ςEbeginning at 2 h after the start of sporulation. TheyrbApromoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intactyrbAmutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination ofyrbAspores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of theyrbAmutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.
Identification and Characterization ofhupT, a Gene Involved in Negative Regulation of Hydrogen Oxidation in Bradyrhizobium japonicum
Authors: Van Soom C. , Lerouge I. , Vanderleyden J. , Ruiz-Argüeso T. , Palacios J. M.
Published: December 2019

TheBradyrhizobium japonicum hupTgene was sequenced, and its gene product was found to be homologous to NtrB-like histidine kinases. AhupTmutant expresses higher levels of hydrogenase activity than the wild-type strain under hydrogenase-inducing conditions (i.e., microaerobiosis plus hydrogen, or symbiosis), whereas in noninducedhupTcells,hupSLexpression is derepressed but does not lead to hydrogenase activity. We conclude that HupT is involved in the repression of HupSL synthesis at the transcriptional level but that enzymatic activation requires inducing conditions.
Combined Genetic and Physical Map of the Complex Genome of
Agrobacterium tumefaciens
Authors: Goodner Brad W. , Markelz Brian P. , Flanagan M. Casey , Crowell Chris B. , Racette Jodi L. , Schilling Brittany A. , Halfon Leah M. , Mellors J. Scott , Grabowski Gregory
Published: December 2019

A combined genetic and physical map of the
Agrobacterium tumefaciens
A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.
Timing of FtsZ Assembly in
Escherichia coli
Authors: Den Blaauwen Tanneke , Buddelmeijer Nienke , Aarsman Mirjam E. G. , Hameete Cor M. , Nanninga Nanne
Published: December 2019

The timing of the appearance of the FtsZ ring at the future site of division in
Escherichia coli
was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.
Amino Acid-DNA Contacts by RhaS: an AraC Family Transcription Activator
Authors: Bhende Prasanna M. , Egan Susan M.
Published: December 2019

RhaS, an AraC family protein, activates
rhaBAD
transcription by binding to
rhaI
, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with
rhaI
were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of
rhaB-lacZ
activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact
rhaI
. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp −65 and −67 in
rhaI
1
, and that Arg202 contacts −46 and Arg206 contacts −48 in
rhaI
2
. In motif 2, we found that Asp250 and Asn252 both contact the bp −79 in
rhaI
1
. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.
Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of
Escherichia coli
: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite
Authors: Lee Angela I. , Delgado Asunción , Gunsalus Robert P.
Published: December 2019

The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using
Escherichia coli
membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 μM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 μM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 μM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.
Identification of a Conserved N-Terminal Sequence Involved in Transmembrane Signal Transduction in EnvZ
Authors: Waukau Jill , Forst Steven
Published: December 2019

To determine whether N-terminal sequences are involved in the transmembrane signaling mechanism of EnvZ, the nucleotide sequences of
envZ
genes from several enteric bacteria were determined. Comparative analysis revealed that the amino acid sequence between Pro41 and Glu53 was highly conserved. To further analyze the role of the conserved sequence,
envZ
of
Escherichia coli
was subjected to random PCR mutagenesis and mutant alleles that produced a high-osmolarity phenotype, in which
ompF
was repressed, were isolated. The mutations identified clustered within, as well as adjacent to, the Pro41-to-Glu53 sequence. These findings suggest that the conserved Pro41-to-Glu53 sequence is involved in the signal transduction mechanism of EnvZ.
Bacterial Conjugation Protein MobA Mediates Integration of Complex DNA Structures into Plant Cells
Authors: Bravo-Angel Ana María , Gloeckler Véronique , Hohn Barbara , Tinland Bruno
Published: December 2019

Agrobacterium tumefaciens
transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by
Agrobacterium
to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5′ end. Isolation and analysis of the most conserved 5′ ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins.
Identification of a Functional
fur
Gene in
Bradyrhizobium japonicum
Authors: Hamza Iqbal , Hassett Richard , O’Brian Mark R.
Published: December 2019

The recent identification of the iron response regulator (Irr) in
Bradyrhizobium japonicum
raised the question of whether the global regulator Fur is present in that organism. A
fur
gene homolog was isolated by the functional complementation of an
Escherichia coli fur
mutant. The
B. japonicum
Fur bound to a Fur box DNA element in vitro, and a
fur
mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus,
B. japonicum
expresses at least two regulators of iron metabolism.
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

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

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

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

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

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

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

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

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

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

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.