bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis, Using P22 Challenge Phage, of the Nitrogen Activator Protein DNA-Binding Site in the
Klebsiella aerogenes put
Operon
Authors: Chen Li-Mei , Goss Thomas J. , Bender Robert A. , Swift Simon , Maloy Stanley
Published: December 2019

The
nac
gene product is a LysR regulatory protein required for nitrogen regulation of several operons from
Klebsiella aerogenes
and
Escherichia coli
. We used P22 challenge phage carrying the
put
control region from
K. aerogenes
to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the
put
operon of
Salmonella typhimurium
, which is not regulated by NAC.
Comparison of the Sensitivities of Two
Escherichia coli
Genes to In Vivo Variation of Lrp Concentration
Authors: Chen ChangFeng , Newman E. B.
Published: December 2019

Transcription of the
Escherichia coli
genes
serA
and
gltBDF
depends on the leucine-responsive regulatory protein, Lrp, and is very much decreased in an
lrp
mutant. By the use of an Lrp-deficient host and the
lrp
gene cloned under a plasmid-borne arabinose pBAD promoter, we varied the amount of Lrp present in the cell and showed that both genes were transcribed in proportion to the amount of Lrp synthesized. The affinity of
serA
for Lrp was four to five times greater than the affinity of
gltD
. Overproduction of Lrp was lethal to the cell.
SacY, a Transcriptional Antiterminator fromBacillus subtilis, Is Regulated by Phosphorylation In Vivo
Authors: Idelson Maria , Amster-Choder Orna
Published: December 2019

SacY antiterminates transcription of thesacBgene inBacillus subtilisin response to the presence of sucrose in the growth medium. We have found that it can substitute for BglG, a homologous protein, in antiterminating transcription of thebgloperon inEscherichia coli. We therefore sought to determine whether, similarly to BglG, SacY is regulated by reversible phosphorylation in response to the availability of the inducing sugar. We show here that two forms of SacY, phosphorylated and nonphosphorylated, exist inB. subtiliscells and that the ratio between them depends on the external level of sucrose. Addition of sucrose to the growth medium after SacY phosphorylation in the cell resulted in its rapid dephosphorylation. The extent of SacY phosphorylation was found to be proportional to the cellular levels of SacX, a putative sucrose permease which was previously shown to have a negative effect on SacY activity. Thus, the mechanism by which thesacsensory system modulatessacBexpression in response to sucrose involves reversible phosphorylation of the regulator SacY, and this process appears to depend on the SacX sucrose sensor. Thesacsystem is therefore a member of the novel family of sensory systems represented bybgl.
Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the
bchI
,
-D
, and
-H
Gene Products of the Green Sulfur Bacterium
Chlorobium vibrioforme
Expressed in
Escherichia coli
Authors: Petersen Bent L. , Jensen Poul Erik , Gibson Lucien C. D. , Stummann Bjarne M. , Hunter C. Neil , Henningsen Knud W.
Published: December 2019

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg
2+
into protoporphyrin IX. Three genes, designated
bchI
,
-D
, and
-H
, from the strictly anaerobic and obligately phototrophic green sulfur bacterium
Chlorobium vibrioforme
show a significant level of homology to the magnesium chelatase-encoding genes
bchI
,
-D
, and
-H
and
chlI
,
-D
, and
-H
of
Rhodobacter sphaeroides
and
Synechocystis
strain PCC6803, respectively. These three genes were expressed in
Escherichia coli
; the subsequent purification of overproduced BchI and -H proteins on an Ni
2+
-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase of
C. vibrioforme
is similar to the magnesium chelatases of the distantly related bacteria
R. sphaeroides
and
Synechocystis
strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the
C. vibrioforme
BchI and -D proteins and the
Synechocystis
strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the
bchI
gene product, were expressed in
E. coli
, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in
C. vibrioforme
.
luxI
- and
luxR
-Homologous Genes of
Rhizobium etli
CNPAF512 Contribute to Synthesis of Autoinducer Molecules and Nodulation of
Phaseolus vulgaris
Authors: Rosemeyer Viola , Michiels Jan , Verreth Christel , Vanderleyden Jos
Published: December 2019

Autoinduction plays an important role in intercellular communication among symbiotic and pathogenic gram-negative bacteria. We report here that a nitrogen-fixing symbiont of
Phaseolus vulgaris
,
Rhizobium etli
CNPAF512, produces at least seven different autoinducer molecules. One of them exhibits a growth-inhibitory effect like that of the bacteriocin
small
[
N
-(3
R
-hydroxy-7-
cis
-tetradecanoyl)-
l
-homoserine lactone]. At least two of the other autoinducers are synthesized by a LuxI-homologous autoinducer synthase. The corresponding
luxI
homologous gene (
raiI
) and a
luxR
homolog (
raiR
) have been identified and characterized. Enhanced expression of
raiI
is dependent on cell density and on the presence of one or more autoinducer molecules synthesized by
R. etli
CNPAF512. A
raiI
mutant was shown to release only three different autoinducer molecules; a
raiR
mutant releases four different autoinducer molecules. Examination of different mutants for nodulation of beans showed that
raiI
is involved in the restriction of nodule number, whereas nitrogen-fixing activity in terms of acetylene reduction per nodule was not affected.
Transcriptional Analysis of the
Bordetella
Alcaligin Siderophore Biosynthesis Operon
Authors: Kang Ho Young , Armstrong Sandra K.
Published: December 2019

The
alc
gene cluster of
Bordetella pertussis
includes three genes,
alcA
,
alcB
, and
alcC
, which are involved in alcaligin siderophore biosynthesis in response to iron starvation. The production of AlcA, AlcB, and AlcC in
Bordetella
cells and the transcriptional organization of
alcA
,
alcB
, and
alcC
were investigated by using a set of three
alc′-′lacZ
gene fusion constructs that were contiguous with the known promoter upstream of
alcA
and extended to fusion junctions within each
alc
cistron. All three
alc′-′lacZ
fusions exhibited iron-repressible reporter gene expression which was abolished by deletion of the 105-bp
alcA
promoter-operator region. In an immunoblot analysis using a monoclonal antibody specific for β-galactosidase, the AlcA-LacZ, AlcB-LacZ, and AlcC-LacZ hybrid proteins were detected in
Bordetella
cells grown under iron-depleted conditions. A
B. pertussis
mutant in which the 105-bp
alcA
promoter-operator region was deleted by allelic exchange was unable to produce detectable levels of siderophore. Hybridization analysis using gene-specific probes showed that
alc
-specific transcript levels in the mutant were negligible compared with those of the wild-type parent. These results confirm that
alcA
,
alcB
, and
alcC
are cotranscribed from an iron-regulated control region immediately upstream of
alcA
. Transcript analysis using hybridization probes representing regions downstream of
alcC
demonstrated that
alc
transcription extends approximately 3.6 kb further downstream from the
alcC
coding region, suggesting the cotranscription of additional, uncharacterized alcaligin system genes.
Both Acetate Kinase and Acetyl Coenzyme A Synthetase Are Involved in Acetate-Stimulated Change in the Direction of Flagellar Rotation in
Escherichia coli
Authors: Barak Rina , Abouhamad Walid N. , Eisenbach Michael
Published: December 2019

Escherichia coli
strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.
Control of
ftsZ
Expression, Cell Division, and Glutamine Metabolism in Luria-Bertani Medium by the Alarmone ppGpp in
Escherichia coli
Authors: Powell Bradford S. , Court Donald L.
Published: December 2019

Inactivation of transcription factor ς
54
, encoded by
rpoN
(
glnF
), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation
ftsZ84
. Mutational defects in three other genes involved in general nitrogen control (
glnD
,
glnG
, and
glnL
) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The
glnL
mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress
ftsZ84
, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the
glnF
null mutation and an elevated copy number of the
relA
gene similarly affect transcription from the upstream (pQ) promoters of the
ftsQAZ
operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of
ftsZ84
by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a
glnF
or
glnD
strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.
Variation in Flagellin Genes and Proteins of
Burkholderia cepacia
Authors: Hales Barbara A. , Morgan J. Alun W. , Hart C. Anthony , Winstanley Craig
Published: December 2019

The majority of isolates of
Burkholderia cepacia
, an important opportunistic pathogen associated with cystic fibrosis, can be classified into two types on the basis of flagellin protein size. Electron microscopic analysis indicates that the flagella of strains with the larger flagellin type (type I) are wider in diameter. Flagellin genes representative of both types were cloned and sequenced to design oligonucleotide primers for PCR amplification of the central variable domain of
B. cepacia
flagellin genes. PCR-restriction fragment length polymorphism analysis of amplified
B. cepacia
flagellin gene products from 16 strains enabled flagellin type classification on the basis of product size and revealed considerable differences in sequence, indicating that the flagellin gene is a useful biomarker for epidemiological and phylogenetic studies of this organism.
The
narA
Locus of
Synechococcus
sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes
Authors: Rubio Luis M. , Flores Enrique , Herrero Antonia
Published: December 2019

The
narA
locus required for nitrate reduction in
Synechococcus
sp. strain PCC 7942 is shown to consist of a cluster of genes, namely,
moeA
,
moaC
,
moaD
,
moaE
, and
moaA
, involved in molybdenum cofactor biosynthesis. The product of the
moaC
gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from
Escherichia coli
and in its C-terminal half to MoaB or Mog. Overexpression of the
Synechococcus moaC
gene in
E. coli
resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of
E. coli
. Insertional inactivation of the
moeA
,
moaC
,
moaE
, and
moaA
genes showed that the
moeA-moa
gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The
moaCDEA
genes constitute an operon which is transcribed divergently from the
moeA
gene. Expression of the
moeA
gene and the
moa
operon was little affected by the nitrogen source present in the culture medium.
The
Yersinia
Yop Virulon: LcrV Is Required for Extrusion of the Translocators YopB and YopD
Authors: Sarker Mahfuzur R. , Neyt Cécile , Stainier Isabelle , Cornelis Guy R.
Published: December 2019

LcrV, an essential piece of the Yop virulon, is encoded by the large
lcrGVsycDyopBD
operon. In spite of repeated efforts, the role of LcrV in the Yop virulon remains elusive. In an attempt to clarify this, we engineered a complete deletion of
lcrV
in the pYV plasmid of
Yersinia enterocolitica
E40 and characterized the phenotype of the mutant. Complementation experiments showed that the mutation was not polar with regard to
yopB
and
yopD
. Nevertheless the mutation abolished secretion of YopB and YopD, while secretion of the other Yops was unaffected or even increased. Northern blot analysis showed that transcription of
yopD
was not affected. YopD could be detected inside the bacteria, showing that the lack of its secretion was not due to a lack of translation or to proteolysis. This indicated that LcrV is specifically involved in the process of release of YopB and YopD. We then investigated the possible interactions between LcrV and YopB or YopD. We constructed a glutathione
S
-transferase–LcrV hybrid protein, and we observed that either YopB or YopD could be copurified with it. The same approach showed that LcrV also interacts with LcrG but not with the chaperone SycD. Using deletants of
lcrV
, we then identified a definite LcrG-binding domain in the C terminus of LcrV.
Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required for
pap
Phase Variation
Authors: Kaltenbach Linda , Braaten Bruce , Tucker Julie , Krabbe Margareta , Low David
Published: December 2019

The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as
ilvIH
, activation of
papBA
transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate
ilvIH
transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on
ilvIH
transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for
pap
and
ilvIH
DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity for
pap
Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-locked
pap
derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on
pap
transcription state.
When an ATPase Is Not an ATPase: at Low Temperatures the C-Terminal Domain of the ABC Transporter CvaB Is a GTPase
Authors: Zhong Xiaotian , Tai Phang C.
Published: December 2019

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from
Escherichia coli
is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg
2+
and Mn
2+
dependent. However, BCTD possesses similar GTPase and ATPase activities at 37°C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10°C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.
An
rne-1 pnp-7
Double Mutation Suppresses the Temperature-Sensitive Defect of
lacZ
Gene Expression in a
divE
Mutant
Authors: Aiso Toshiko , Ohki Reiko
Published: December 2019

A
divE
mutant, which has a temperature-sensitive mutation in the tRNA
1
Ser
gene, exhibits differential loss of the synthesis of certain proteins, such as β-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In
Escherichia coli
, the UCA codon is recognized only by tRNA
1
Ser
. Several genes containing UCA codons are normally expressed after a temperature shift to 42°C in the
divE
mutant. Therefore, it is unlikely that the defect in protein synthesis at 42°C is simply caused by a defect in the decoding function of the mutant tRNA
1
Ser
. In this study, we sought to determine the cause of the defect in
lacZ
gene expression in the
divE
mutant. It has also been shown that the defect in
lacZ
gene expression is accompanied by a decrease in the amount of
lacZ
mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in
lacZ
gene expression, we constructed
divE
mutants containing
rne-1
,
rnb-500
, and
pnp-7
mutations in various combinations. We found that the defect was almost completely restored by introducing an
rne-1 pnp-7
double mutation into the
divE
mutant. Northern hybridization analysis showed that the
rne-1
mutation stabilized
lacZ
mRNA, whereas the
pnp-7
mutation stabilized mutant tRNA
1
Ser
, at 44°C. We present a mechanism that may explain these results.
Balance between Endogenous Superoxide Stress and Antioxidant Defenses
Authors: Gort Amy Strohmeier , Imlay James A.
Published: December 2019

Cells devoid of cytosolic superoxide dismutase (SOD) suffer enzyme inactivation, growth deficiencies, and DNA damage. It has been proposed that the scant superoxide (O
2
−
) generated by aerobic metabolism harms even cells that contain abundant SOD. However, this idea has been difficult to test. To determine the amount of O
2
−
that is needed to cause these defects, we modulated the O
2
−
concentration inside
Escherichia coli
by controlling the expression of SOD. An increase in O
2
−
of more than twofold above wild-type levels substantially diminished the activity of labile dehydratases, an increase in O
2
−
of any more than fourfold measurably impaired growth, and a fivefold increase in O
2
−
sensitized cells to DNA damage. These results indicate that
E. coli
constitutively synthesizes just enough SOD to defend biomolecules against endogenous O
2
−
so that modest increases in O
2
−
concentration diminish cell fitness. This conclusion is in excellent agreement with quantitative predictions based upon previously determined rates of intracellular O
2
−
production, O
2
−
dismutation, dehydratase inactivation, and enzyme repair. The vulnerability of bacteria to increased intracellular O
2
−
explains the widespread use of superoxide-producing drugs as bactericidal weapons in nature.
E. coli
responds to such drugs by inducing the SoxRS regulon, which positively regulates synthesis of SOD and other defensive proteins. However, even toxic amounts of endogenous O
2
−
did not activate SoxR, and SoxR activation by paraquat was not at all inhibited by excess SOD. Therefore, in responding to redox-cycling drugs, SoxR senses some signal other than O
2
−
.
Unusual Organization of the Genes Coding for HydSL, the Stable [NiFe]Hydrogenase in the Photosynthetic Bacterium
Thiocapsa roseopersicina
BBS
Authors: Rakhely Gabor , Colbeau Annette , Garin Jerome , Vignais Paulette M. , Kovacs Kornel L.
Published: December 2019

The characterization of a
hyd
gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in
Thiocapsa roseopersicina
BBS, a purple sulfur photosynthetic bacterium belonging to the family
Chromatiaceae
, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947–951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567–3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes
hydS
and
hydL
, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the
hmc
operon, coding for a transmembrane electron transfer complex in
Desulfovibrio vulgaris
. Other accessory proteins are not found immediately downstream or upstream of
hydSL
. A
hup
gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in
T. roseopersicina
was reported earlier (A. Colbeau et al. Gene 140:25–31, 1994). The deduced amino acid sequences of the two small (
hupS
and
hydS
) and large subunit (
hupL
and
hydL
) sequences share 46 and 58% identity, respectively. The
hup
and
hyd
genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by
hydSL
, and the inducible and unstable second hydrogenase may be the product of the
hup
gene cluster.
In Vivo Protein Interactions within the
Escherichia coli
DNA Polymerase III Core
Authors: Jonczyk Piotr , Nowicka Adrianna , Fijałkowska Iwona J. , Schaaper Roel M. , Cieśla Zygmunt
Published: December 2019

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of
Escherichia coli
could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the
Saccharomyces cerevisiae
two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the
mutD5
and
dnaQ926
mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, the
dnaQ49
mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of
dnaQ49
may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.
Molecular Cloning, Expression, and Characterization of the Genes Encoding the Two Essential Protein Components of
Micrococcus luteus
B-P 26 Hexaprenyl Diphosphate Synthase
Authors: Shimizu Naoto , Koyama Tanetoshi , Ogura Kyozo
Published: December 2019

The structural genes encoding the two essential components A and B of hexaprenyl diphosphate synthase, which produce the precursor of the prenyl side chain of menaquinone-6, were cloned from
Micrococcus luteus
B-P 26.
Isolation and Purification of Two Novel Streptomycete RNase Inhibitors, SaI14 and SaI20, and Cloning, Sequencing, and Expression inEscherichia coliof the Gene Coding for SaI14
Authors: Krajcikova Daniela , Hartley Robert W. , Sevcik Jozef
Published: December 2019

Two new RNase inhibitors, SaI14 (Mr, ∼14,000) and SaI20 (Mr, ∼20,000), were isolated and purified from aStreptomyces aureofaciensstrain. The genesai14, coding for SaI14 protein, was cloned and expressed inEscherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor fromBacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.
The Folate Branch of the Methionine Biosynthesis Pathway in
Streptomyces lividans
: Disruption of the 5,10-Methylenetetrahydrofolate Reductase Gene Leads to Methionine Auxotrophy
Authors: Blanco Jorge , Coque Juan Jose R. , Martin Juan F.
Published: December 2019

In enterobacteria, the methyl group of methionine is donated by 5-methyltetrahydrofolate that is synthesized from
N
5,10
-methylenetetrahydrofolate by the 5,10-methylenetetrahydrofolate reductase. The
Streptomyces lividans metF
gene, which encodes 5,10-methylenetetrahydrofolate reductase, has been cloned. It encodes a protein of 307 amino acids with a deduced molecular mass of 33,271 Da. S1 exonuclease mapping of the transcription initiation site showed that the
metF
gene is expressed, forming a leaderless mRNA. A 13-bp tandem repeat located immediately upstream of the promoter region shows homology with the consensus MetR-binding sequence of
Salmonella typhimurium
. Expression of
metF
in multicopy plasmids in
S. lividans
resulted in accumulation of a 32-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Disruption of the
metF
gene led to methionine auxotrophy. Integration of the disrupting plasmid at the
metF
locus was confirmed by Southern hybridization in three randomly isolated transformants. The methionine auxotrophy was complemented by transformation of the auxotrophs with an undisrupted
metF
gene. These results indicate that the folate branch is essential for methionine biosynthesis in streptomycetes, as occurs in enterobacteria.
Cloning and Sequencing of a Form II Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from the Bacterial Symbiont of the Hydrothermal Vent Tubeworm
Riftia pachyptila
Authors: Robinson Jonathan J. , Stein Jeffrey L. , Cavanaugh Colleen M.
Published: December 2019

The bacterial symbiont of the hydrothermal vent tubeworm fixes carbon via the Calvin-Benson cycle and has been shown previously to express a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene
cbbM
, which encodes this enzyme, has been cloned and sequenced. The gene has the highest identity with the
cbbM
gene from
Rhodospirillum rubrum
, and analysis of the inferred amino acid sequence reveals that all active-site residues are conserved. This is the first form II RubisCO cloned and sequenced from a chemoautotrophic symbiont and from a deep-sea organism.
The
ars
Operon in the
skin
Element of
Bacillus subtilis
Confers Resistance to Arsenate and Arsenite
Authors: Sato Tsutomu , Kobayashi Yasuo
Published: December 2019

The
Bacillus subtilis skin
element confers resistance to arsenate and arsenite. The
ars
operon in the
skin
element contains four genes in the order
arsR
,
ORF2
,
arsB
, and
arsC
. Three of these genes are homologous to the
arsR
,
arsB
, and
arsC
genes from the staphylococcal plasmid pI258, while no homologs of
ORF2
have been found. Inactivation of
arsR
,
arsB
, or
arsC
results in either constitutive expression of
ars
, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the
ars
operon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length
ars
transcript of about 2.4 kb was induced by arsenate and that the
ars
promoter contains sequences that resemble the −10 and −35 regions of promoters that are recognized by Eς
A
.
Involvement of
recF
,
recO
, and
recR
Genes in UV-Radiation Mutagenesis of
Escherichia coli
Authors: Liu Ying-Hsiu , Cheng Ann-Joy , Wang Tzu-chien V.
Published: December 2019

The
recF
,
recO
, and
recR
genes were originally identified as those affecting the RecF pathway of recombination in
Escherichia coli
cells. Several lines of evidence suggest that the
recF
,
recO
, and
recR
genes function at the same step of recombination and postreplication repair. In this work, we report that null mutations in
recF
,
recO
, or
recR
greatly reduce UV-radiation mutagenesis (UVM) in an assay for reversion from a Trp
−
(
trpE65
) to a Trp
+
phenotypes. Introduction of the defective
lexA51
mutation [
lexA51
(Def)] and/or UmuD′ into
recF
,
recO
, and
recR
mutants failed to restore normal UVM in the mutants. On the other hand, the presence of
recA2020
, a suppressor mutation for
recF
,
recO
, and
recR
mutations, restored normal UVM in
recF
,
recO
, and
recR
mutants. These results indicate an involvement of the
recF
,
recO
, and
recR
genes and their products in UVM, possibly by affecting the third role of RecA in UVM.
Probing the Yeast Phase-Specific Expression of the
CBP1
Gene in
Histoplasma capsulatum
Authors: Patel Jean Baldus , Batanghari Janet West , Goldman William E.
Published: December 2019

Histoplasma capsulatum
is a pathogenic fungus that exists in two distinct forms. The saprophytic mycelial phase inhabits moist soil environments; once inhaled, hyphae and conidia convert to a unicellular yeast phase that is capable of parasitizing macrophage phagolysosomes. Yeasts cultures, but not mycelial cultures, release large quantities of a calcium-binding protein (CBP) which may be important in calcium acquisition during intracellular parasitism. In this study, we show that the gene encoding CBP (
CBP1
) is transcriptionally regulated. To identify promoter sequences that are important for yeast phase-specific activity, we created a series of fusions between successively truncated
CBP1
5′ untranslated regulatory sequences and the
Eschericha coli lacZ
gene. The fusions were constructed on a telomeric shuttle plasmid that can replicate autonomously in the fungus. By assaying for β-galactosidase activity from
H. capsulatum
transformants, we identified a 102-bp region that mediates promoter activation and yeast phase promoter activity. Base pair substitution analysis suggests that the sequences between 839 and 877 bp upstream of the start codon are the most important for this positive regulation.
The Methyl Group of the
N
6
-Methyl-
N
6
-Threonylcarbamoyladenosine in tRNA of
Escherichia coli
Modestly Improves the Efficiency of the tRNA
Authors: Qian Qiang , Curran James F. , Björk Glenn R.
Published: December 2019

tRNA species that read codons starting with adenosine (A) contain
N
6
-threonylcarbamoyladenosine (t
6
A) derivatives adjacent to and 3′ of the anticodons from all organisms. In
Escherichia coli
there are 12 such tRNA species of which two (tRNA
GGU
Thr1
and tRNA
GGU
Thr3
) have the t
6
A derivative
N
6
-methyl-
N
6
-threonylcarbamoyladenosine (m
6
t
6
A37). We have isolated a mutant of
E. coli
that lacks the m
6
t
6
A37 in these two tRNA
GGU
Thr
species. These tRNA species in the mutant are likely to have t
6
A37 instead of m
6
t
6
A37. We show that the methyl group of m
6
t
6
A37 originates from
S
-adenosyl-
l
-methionine and that the gene (
tsaA
) which most likely encodes tRNA(m
6
t
6
A37)methyltransferase is located at min 4.6 on the
E. coli
chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA
GGU
Thr
to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the
tsaA1
mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m
6
t
6
A37-containing tRNA
GGU
Thr
species. We show that the
tsaA1
mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m
6
t
6
A37 in tRNA
GGU
Thr
slightly reduces the efficiency of this tRNA to read cognate codon ACC.
The
Escherichia coli mrsC
Gene Is Required for Cell Growth and mRNA Decay
Authors: Granger Laurie L. , O’Hara Eileen B. , Wang Rong-Fu , Meffen Frances V. , Armstrong Katherine , Yancey Stephanie D. , Babitzke Paul , Kushner Sidney R.
Published: December 2019

We have identified a gene in
Escherichia coli
that is required for both the normal decay of mRNA and RNA synthesis. Originally designated
mrsC
(mRNA stability), the
mrsC505
mutation described here is, in fact, an allele of the
hflB/ftsH
locus (R.-F. Wang et al., J. Bacteriol. 180:1929–1938, 1998). Strains carrying the thermosensitive
mrsC505
allele stopped growing soon after the temperature was shifted to 44°C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44°C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the
mrsC505
single mutant. In an
rne-1 mrsC505
double mutant, the average half-life was 19.8 min. Inactivating
mrsC
significantly increased the half-lives of the
trxA
,
cat
,
secG
, and
kan
mRNAs, particularly in an
mrsC505 pnp-7 rnb-500 rne-1
multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of
mrsC505
multiple mutants at 44°C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.
Levels of the Vsr Endonuclease Do Not Regulate Stationary-Phase Reversion of a Lac
−
Frameshift Allele in
Escherichia coli
Authors: Foster Patricia L. , Rosche William A.
Published: December 2019

Vsr endonuclease, which initiates very short patch repair, has been hypothesized to regulate mutation in stationary-phase cells. Overexpression of Vsr does dramatically increase the stationary-phase reversion of a Lac
−
frameshift allele, but the absence of Vsr has no effect. Thus, at least in this case, Vsr has no regulatory role in stationary-phase mutation, and the effects of Vsr overproduction are likely to be artifactual.
Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in
Escherichia coli
Authors: Murphy Kenan C.
Published: December 2019

Replacement of
Escherichia coli
’s RecBCD function with phage λ’s Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a
recBC sbcBC
or
recD
strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The Δ
recBCD
::P

lac

-red kan
replacement allele can be P1 transduced to other
E. coli
strains, making the hyper-Rec phenotype easily transferable.
Isolation and Characterization of
EPD1
, an Essential Gene for Pseudohyphal Growth of a Dimorphic Yeast,
Candida maltosa
Authors: Nakazawa Takanobu , Horiuchi Hiroyuki , Ohta Akinori , Takagi Masamichi
Published: December 2019

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast,
Candida maltosa
(T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030–5036, 1997). To understand the mechanism of this transition, we screened the gene library of
C. maltosa
for sequences which could suppress this morphological change. As a result, we isolated the 5′ end of a new gene,
EPD1
(for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epd1p was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of
Saccharomyces cerevisiae
, and Phr1p and Phr2p of
Candida albicans
. The expression of
EPD1
was moderately regulated by environmental pH. A homozygous
EPD1
null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble β-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by
n
-hexadecane at pH 4 or pH 7, suggesting that
EPD1
is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the
GAL
promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length
EPD1
did not. This result and the initial isolation of the 5′ end of
EPD1
as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.
IS
6110
Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related
Mycobacterium tuberculosis
Strains
Authors: Fang Z. , Morrison N. , Watt B. , Doig C. , Forbes K. J.
Published: December 2019

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the
Mycobacterium tuberculosis
genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS
6110
, and IS
1081
. These, especially IS
6110
and the DR locus, have been widely used as genetic markers to differentiate
M. tuberculosis
isolates and will continue to be so used, due to the conserved nature of the genome of
M. tuberculosis
. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of
M. tuberculosis
. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS
6110
transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among
M. tuberculosis
strains. Our results suggest that IS
6110
restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS
6110
, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS
6110
copy in the DR locus was noted, and the mechanisms of this loss are discussed.
Transformation of
Rickettsia prowazekii
to Rifampin Resistance
Authors: Rachek Lyudmila I. , Tucker Aimee M. , Winkler Herbert H. , Wood David O.
Published: December 2019

Rickettsia prowazekii
, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial
rpoB
gene that confers resistance to rifampin and used it to demonstrate allelic exchange in
R. prowazekii
. Comparison of the
rpoB
sequences from the rifampin-sensitive (Rif
s
) Madrid E strain and a rifampin-resistant (Rif
r
) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the
R. prowazekii
RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rif
s
Madrid E strain of
R. prowazekii
by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rif
r
region of rickettsial
rpoB
. This is the first successful demonstration of genetic transformation of
Rickettsia prowazekii
and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.
Visualization of Penicillin-Binding Proteins during Sporulation of
Streptomyces griseus
Authors: Hao Jiang , Kendrick Kathleen E.
Published: December 2019

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of
Streptomyces griseus
. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of
S. griseus.
A Novel DNA Polymerase Family Found inArchaea
Authors: Ishino Yoshizumi , Komori Kayoko , Cann Isaac K. O. , Koga Yosuke
Published: December 2019

One of the most puzzling results from the complete genome sequence of the methanogenic archaeonMethanococcus jannaschiiwas that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes inM. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosushave been located and cloned. The gene products ofM. jannaschiiexpressed inEscherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.
The
tfdK
Gene Product Facilitates Uptake of 2,4-Dichlorophenoxyacetate by
Ralstonia eutropha
JMP134(pJP4)
Authors: Leveau Johan H. J. , Zehnder Alexander J. B. , van der Meer Jan Roelof
Published: December 2019

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by
Ralstonia eutropha
JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated
tfdK
, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the
tfdK
gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A
tfdA
mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.
Erwinia amylovora
Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, through the Hrp (Type III Secretion) Pathway
Authors: Bogdanove Adam J. , Bauer David W. , Beer Steven V.
Published: December 2019

Erwinia amylovora
was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of
Pseudomonas syringae
pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an
hrp
gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.
S-Layered
Aneurinibacillus
and
Bacillus
spp. Are Susceptible to the Lytic Action of
Pseudomonas aeruginosa
Membrane Vesicles
Authors: Kadurugamuwa J. L. , Mayer A. , Messner P. , Sára M. , Sleytr U. B. , Beveridge T. J.
Published: December 2019

When S-layered strains of
Bacillus stearothermophilus
and
Aneurinibacillus thermoaerophilus
, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from
P. aeruginosa
during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact.
meso
-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.
Control of 5′,5′-Dinucleoside Triphosphate Catabolism by
APH1
, a
Saccharomyces cerevisiae
Analog of Human
FHIT
Authors: Chen Josiane , Brevet Annie , Blanquet Sylvain , Plateau Pierre
Published: December 2019

The putative human tumor suppressor gene
FHIT
(fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P
1
,P
3
-triphosphate (Ap
3
A) hydrolase. In this report, we show that the
Saccharomyces cerevisiae APH1
gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap
3
A being the preferred substrate. Accordingly, disruption of the
APH1
gene produced viable
S. cerevisiae
cells containing reduced Ap
3
A-hydrolyzing activity and a 30-fold-elevated Ap
3
N concentration.