bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate Ambiguity of 3-Deoxy-
d
-
manno
-Octulosonate 8-Phosphate Synthase from
Neisseria gonorrhoeae
in the Context of Its Membership in a Protein Family Containing a Subset of 3-Deoxy-
d
-
arabino
-Heptulosonate 7-Phosphate Synthases
Authors: Subramaniam Prem S. , Xie Gang , Xia Tianhui , Jensen Roy A.
Published: December 2019

3-Deoxy-
d
-
manno
-octulosonate 8-phosphate (KDOP) synthase and 3-deoxy-
d
-
arabino
-heptulosonate 7-phosphate (DAHP) synthase catalyze similar phosphoenolpyruvate-utilizing reactions. The genome of
Neisseria gonorrhoeae
contains one gene encoding KDOP synthase and one gene encoding DAHP synthase. Of the two nonhomologous DAHP synthase families known, the
N. gonorrhoeae
protein belongs to the family I assemblage. KDOP synthase exhibited an ability to replace arabinose-5-P with either erythrose-4-P or ribose-5-P as alternative substrates. The results of periodate oxidation studies suggested that the product formed by KDOP synthase with erythrose-4-P as the substrate was 3-deoxy-
d
-
ribo
-heptulosonate 7-P, an isomer of DAHP. As expected, this product was not utilized as a substrate by dehydroquinate synthase. The significance of the ability of KDOP synthase to substitute erythrose-4-P for arabinose-5-P is (i) recognition of the possibility that the KDOP synthase might otherwise be mistaken for a species of DAHP synthase and (ii) the possibility that the broad-specificity type of KDOP synthase might be a relatively vulnerable target for antimicrobial agents which mimic the normal substrates. An analysis of sequences in the database indicates that the family I group of DAHP synthase has a previously unrecognized membership which includes the KDOP synthases. The KDOP synthases fall into a subfamily grouping which includes a small group of DAHP synthases. Thus, family I DAHP synthases separate into two subfamilies, one of which includes the KDOP synthases. The two subfamilies appear to have diverged prior to the acquisition of allosteric-control mechanisms for DAHP synthases. These allosteric control specificities are highly diverse and correlate with the presence of N-terminal extensions which lack homology with one another.
Identification and Characterization of
aarF
, a Locus Required for Production of Ubiquinone in
Providencia stuartii
and
Escherichia coli
and for Expression of 2′-
N
-Acetyltransferase in
P. stuartii
Authors: Macinga David R. , Cook Gregory M. , Poole Robert K. , Rather Philip N.
Published: December 2019

Providencia stuartii
contains a chromosomal 2′-
N
-acetyltransferase [AAC(2′)-Ia] involved in the O acetylation of peptidoglycan. The AAC(2′)-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in
P. stuartii
, designated
aarF
, that is required for the expression of AAC(2′)-Ia. Northern (RNA) analysis demonstrated that
aac(2′)-Ia
mRNA levels were dramatically decreased in a
P. stuartii
strain carrying an
aarF
::Cm disruption. The
aarF
::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The
aarF
locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the
Escherichia coli
chromosome. An
E. coli yigR
::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the
aarF
and the
E. coli yigQR
loci were functionally equivalent. The
aarF
or
yigQR
genes were unable to complement
ubiD
and
ubiE
mutations that are also present at 86 min on the
E. coli
chromosome. This result indicates that
aarF
(
yigQR
) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.
Diacylglycerol Kinase Is Involved in Regulation of Expression of the Lantibiotic Mutacin II of
Streptococcus mutans
Authors: Chen Ping , Novak Jan , Qi Fengxia , Caufield Page W.
Published: December 2019

Genetic characterization of a Tn
916
transposon mutant,
Streptococcus mutans
T8-1, defective in mutacin II production, revealed that the transposon was inserted into the 3′ region of a diacylglycerol kinase (
dgk
) gene. The insertion occurred in the same region as described for another
S. mutans
mutant, GS5Tn1, which was altered in its ability to respond to environmental stress (Y. Yamashita, T. Takehara, and H. K. Kuramitsu, J. Bacteriol. 175:6220–6228, 1993). Quantitative primer extension from the mutacin structural gene
mutA
showed a decreased level (about eightfold) of
mutA
transcription for mutant T8-1. Mutacin production was restored by transforming mutant T8-1 with integration vector pVA891 containing an intact
dgk
gene. These data indicated that the full-length
dgk
gene product along with the mutacin biosynthetic operon are required for the production of the mutacin II lantibiotic.
Ammonification in
Bacillus subtilis
Utilizing Dissimilatory Nitrite Reductase Is Dependent on
resDE
Authors: Hoffmann Tamara , Frankenberg Nicole , Marino Marco , Jahn Dieter
Published: December 2019

During anaerobic nitrate respiration
Bacillus subtilis
reduces nitrate via nitrite to ammonia. No denitrification products were observed.
B. subtilis
wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by
resDE
. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.
The Capsule and S-Layer: Two Independent and Yet Compatible Macromolecular Structures in
Bacillus anthracis
Authors: Mesnage Stéphane , Tosi-Couture Evelyne , Gounon Pierre , Mock Michèle , Fouet Agnès
Published: December 2019

Bacillus anthracis
, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the
B. anthracis
surface layer (S-layer). The
B. anthracis
paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-γ-
d
-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.
Role of the Cold-Box Region in the 5′ Untranslated Region of the
cspA
mRNA in Its Transient Expression at Low Temperature in
Escherichia coli
Authors: Fang Li , Hou Yan , Inouye Masayori
Published: December 2019

Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced in
Escherichia coli
. However, when the 5′ untranslated region (5′ UTR) of
cspA
mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the “cold box” existing in the 5′ UTRs of
cspA
,
cspB
, and
csdA
. Here, we demonstrate that the overproduction of the 5′ UTR of not only
cspA
but also
cspB
and
csdA
mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the
cspA
5′ UTR its derepression function was abolished. The amount of mRNA from the chromosomal
cspA
gene was much higher in cells overproducing the wild-type 5′ UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5′ UTR. The stability of the chromosomal
cspA
mRNA in cells overproducing the wild-type 5′ UTR was almost identical to that in cells overproducing the cold-box-deleted 5′ UTR. Therefore, the derepression of
cspA
caused by overproduction of 5′ UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in
cspA
transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with a
cspA
mutant strain containing a cold-box-deleted
cspA
gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.
Roles of DctA and DctB in Signal Detection by the Dicarboxylic Acid Transport System of
Rhizobium leguminosarum
Authors: Reid Colm J. , Poole Philip S.
Published: December 2019

The
dctA
gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for
dctB
and
dctD
. The
dctA
promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader.
dctAp
also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in the
dctA-dctB
intergenic region (
dctA101
) which locks transcription of
dctA
at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix
−
) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation of
dctB
or
dctD
in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.
Unconventional Genomic Organization in the Alpha Subgroup of the
Proteobacteria
Authors: Jumas-Bilak Estelle , Michaux-Charachon Sylvie , Bourg Gisele , Ramuz Michel , Allardet-Servent Annick
Published: December 2019

Pulsed-field gel electrophoresis was used to analyze the genomic organization of 16 bacteria belonging or related to the family
Rhizobiaceae
of the alpha subgroup of the class
Proteobacteria
. The number and sizes of replicons were determined by separating nondigested DNA. Hybridization of an
rrn
gene probe was used to distinguish between chromosomes and plasmids. Members of the genus
Agrobacterium
all possess two chromosomes, and each biovar has a specific genome size. As previously demonstrated for
Agrobacterium tumefaciens
C58, the smaller chromosomes of
Agrobacterium
biovar 1 and
Agrobacterium rubi
strains appear to be linear. The genomes of
Rhizobium
strains were all of similar sizes but were seen to contain either one, two, or three megareplicons. Only one chromosome was present in the member of the related genus
Phyllobacterium
. We found one or two chromosomes in
Rhodobacter
and
Brucella
species, two chromosomes in
Ochrobactrum anthropi
, and one chromosome in
Mycoplana dimorpha
and
Bartonella quintana
; all of these genera are related to the
Rhizobiaceae
. The presence of multiple chromosomes is discussed from a phylogenetic and taxonomic point of view.
Suppressor Scanning at Positions 177 and 236 in the
Escherichia coli
Lactose/H
+
Cotransporter and Stereotypical Effects of Acidic Substituents That Suggest a Favored Orientation of Transmembrane Segments Relative to the Lipid Bilayer
Authors: King Steven C. , Li Suzhen
Published: December 2019

Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents. Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.
Genes Essential for Nod Factor Production and Nodulation Are Located on a Symbiotic Amplicon (AMP
Rtr
CFN299pc60) in
Rhizobium tropici
Authors: Mavingui Patrick , Laeremans Toon , Flores Margarita , Romero David , Martínez-Romero Esperanza , Palacios Rafael
Published: December 2019

Amplifiable DNA regions (amplicons) have been identified in the genome of
Rhizobium etli
. Here we report the isolation and molecular characterization of a symbiotic amplicon of
Rhizobium tropici
. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of
R. tropici
CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into
Escherichia coli
and then into
Agrobacterium tumefaciens
. The 60-kb symbiotic amplicon, which we termed AMP
Rtr
CFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence IS
Rtr1
. Amplification of AMP
Rtr
CFN299pc60 through homologous recombination between IS
Rtr1
repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless
A. tumefaciens
strain confers on the transconjugant the ability to produce
R. tropici
Nod factors and to nodulate
Phaseolus vulgaris
, indicating that
R. tropici
genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.
Posttranscriptional Modifications in 16S and 23S rRNAs of the Archaeal Hyperthermophile
Sulfolobus solfataricus
Authors: Noon Kathleen R. , Bruenger Eveline , McCloskey James A.
Published: December 2019

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile
Sulfolobus solfactaricus
by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75°C, compared with 11 and 23 sites, respectively, in
Escherichia coli
. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2′ of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83°C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23°C range shown for 2′-
O
-methyladenosine in 16S rRNA (21% increase) and for 2′-
O
-methylcytidine (24%) and 2′-
O
-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.
Evidence for a Conjugation-Like Mechanism of DNA Transfer in
Helicobacter pylori
Authors: Kuipers Ernst J. , Israel Dawn A. , Kusters Johannes G. , Blaser Martin J.
Published: December 2019

Many strains of
Helicobacter pylori
are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among
H. pylori
strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with
H. pylori
cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between
H. pylori
cells.
DNA Synthesis and Viability of a
mutT
Derivative of
Escherichia coli
WP2 under Conditions of Amino Acid Starvation and Relation to Stationary-Phase (Adaptive) Mutation
Authors: Bridges Bryn A. , Ereira Sharon
Published: December 2019

Escherichia coli
WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10
8
on plates lacking the required amino acid. They lose viability for some days, and then a subpopulation recovers and there is cell turnover. At very low plating densities (around 10
2
per plate), almost every cell will eventually form a small but visible colony. At intermediate plating densities (10
6
to 10
7
per plate), there is an immediate increase in the number of viable bacteria. The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species. Mutations in
mutT
bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10
7
per plate). We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10
8
scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid. The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a
polA
derivative. We cannot exclude the possibility that this residual DNA synthesis is peculiar to
mutT
bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain. If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10
7
per plate) and reduced the rate of appearance of mutants at plating densities above 10
8
per plate.
glkA
Is Involved in Glucose Repression of Chitinase Production in
Streptomyces lividans
Authors: Saito Akihiro , Fujii Takeshi , Yoneyama Tadakatsu , Miyashita Kiyotaka
Published: December 2019

Chitinase production in
Streptomyces lividans
is induced by chitin and repressed in the presence of glucose. A mutant of
S. lividans
TK24, strain G015, which was defective in glucose repression of chitinase production, was obtained by screening colonies for zones of clearing on colloidal chitin agar plates containing 1.0% (wt/vol) glucose. The transcriptional analysis of
chiA
in G015 with
xylE
, which encodes catechol 2,3-dioxygenase, as a reporter gene showed that the transcription from the
chiA
promoter of
S. lividans
TK24 occurred regardless of the presence of glucose. G015 was resistant to 2-deoxyglucose (2-DOG) and did not utilize glucose as a sole carbon source. When a DNA fragment containing
glkA
, a gene for glucose kinase, of
Streptomyces coelicolor
A3(2) was introduced into strain G015 on a low-copy-number plasmid, the sensitivity to 2-DOG, the ability to utilize glucose, and the glucose repression of chitinase production were restored. These results indicate that
glkA
is involved in glucose repression of chitinase production in
S. lividans
TK24.
Translation of the Flagellar Gene
fliO
of
Salmonella typhimurium
from Putative Tandem Starts
Authors: Schoenhals Gary J. , Kihara May , Macnab Robert M.
Published: December 2019

The flagellar gene
fliO
of
Salmonella typhimurium
can be translated from an AUG codon that overlaps the termination codon of
fliN
(K. Ohnishi et al., J. Bacteriol. 179:6092–6099, 1997). However, it had been concluded on the basis of complementation analysis that in
Escherichia coli
a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189–197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene
cheA
; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with
cheA
. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in
S. typhimurium
there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A
fliO
null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between
S. typhimurium
and
E. coli
and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.
Mutational Analysis of the TnrA-Binding Sites in the
Bacillus subtilis nrgAB
and
gabP
Promoter Regions
Authors: Wray Lewis V. , Zalieckas Jill M. , Ferson Amy E. , Fisher Susan H.
Published: December 2019

Transcription of the
Bacillus subtilis nrgAB
promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN
7
TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated
nrgAB
,
gabP
P2, and
nas
promoters. Oligonucleotide-directed mutagenesis of the
nrgAB
promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of
nrgAB
expression. Mutations that alter the relative distance between the two half-sites of the
nrgAB
TnrA site abolish nitrogen regulation of
nrgAB
expression. Spacer mutations that change the relative distance between the TnrA site and −35 region of the
nrgAB
promoter reveal that activation of
nrgAB
expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the
gabP
P2 TnrA site showed that this sequence is also required for nitrogen-regulated
gabP
P2 expression. The TnrA protein, expressed in an overproducing
Escherichia coli
strain, had a 625-fold-higher affinity for the wild-type
nrgAB
promoter DNA than for a mutated
nrgAB
promoter DNA fragment that is unable to activate
nrgAB
expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate
nrgAB
expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.
Replicase, Excisionase, and Integrase Genes of the
Streptomyces
Element pSAM2 Constitute an Operon Positively Regulated by the
pra
Gene
Authors: Sezonov Guennadi , Duchêne Anne-Marie , Friedmann Annick , Guérineau Michel , Pernodet Jean-Luc
Published: December 2019

pSAM2 is a site-specific integrative element from
Streptomyces ambofaciens
. The
pra
gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the
pra
gene was placed under the control of the inducible
tipAp
promoter. Transformation of
Streptomyces lividans
by these derivatives was efficient only when
pra
expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the
pra
expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes
repSA
,
xis
, and
int
coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by
pra.
Promoter Region of the
Escherichia coli
O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence
Authors: Marolda Cristina L. , Valvano Miguel A.
Published: December 2019

We report the identification of the promoter region of the
Escherichia coli
O7-specific lipopolysaccharide (LPS) gene cluster (
wb
EcO7
). Typical −10 and −35 sequences were found to be located in the intervening region between
galF
and
rlmB
, the first gene of the
wb
EcO7
cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two
ops
sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless
lac
operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the
ops
sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the
waaQGPSBIJYZK
genes. We observed another predicted stem-loop, located immediately downstream from the
wb
EcO7
transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes.
wb
EcO7
-
lac
transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg
2+
concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the
wb
EcO7
promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.
Folding-Based Suppression of Extracytoplasmic Toxicity Conferred by Processing-Defective LamB
Authors: Cosma Christine L. , Crotwell Michelle D. , Burrows Stephanie Y. , Silhavy Thomas J.
Published: December 2019

We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of
Escherichia coli
. Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase. As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane. These mutant porins are toxic when secreted to the cell envelope. Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J. H. Carlson and T. J. Silhavy, J. Bacteriol. 175:3327–3334, 1993). We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism. Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane. Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA. The suppression mediated by TetA′ is independent of the CpxA/CpxR regulon and the ς
E
regulon, both of which are involved in regulating protein trafficking functions in the cell envelope.
Anaerobic Growth, a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles
Authors: Ramírez-Arcos Sandra , Fernández-Herrero Luis A. , Marín Irma , Berenguer José
Published: December 2019

Despite the fact that the extreme thermophilic bacteria belonging to the genusThermusare classified as strict aerobes, we have shown thatThermus thermophilusHB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramı́rez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that thisnaroperon can be transferred by conjugation to an aerobicThermusstrain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives ofEscherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage betweennarand a conjugative plasmid integrated into the chromosome. First, thenaroperon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of thenarcluster. Finally, the mating time required for the transfer of thenaroperon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream ofnar.
The ms
2
io
6
A37 Modification of tRNA in
Salmonella typhimurium
Regulates Growth on Citric Acid Cycle Intermediates
Authors: Persson Britt C. , Ólafsson Ólafur , Lundgren Hans K. , Hederstedt Lars , Björk Glenn R.
Published: December 2019

The modified nucleoside 2-methylthio-
N
-6-isopentenyl adenosine (ms
2
i
6
A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA
I,V
Ser
in
Escherichia coli
. In
Salmonella typhimurium
, 2-methylthio-
N
-6-(
cis
-hydroxy)isopentenyl adenosine (ms
2
io
6
A; also referred to as 2-methylthio
cis
-ribozeatin) is found in tRNA, most likely in the species that have ms
2
i
6
A in
E. coli
. Mutants (
miaE
) of
S. typhimurium
in which ms
2
i
6
A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of
S. typhimurium
to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms
2
io
6
A37 and is not due to a second cellular function of tRNA (ms
2
io
6
A37)hydroxylase, the
miaE
gene product. We suggest that
S. typhimurium
senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.
Streptomyces griseus
Protease B: Secretion Correlates with the Length of the Propeptide
Authors: Baardsnes J. , Sidhu S. , MacLeod A. , Elliott J. , Morden D. , Watson J. , Borgford T.
Published: December 2019

Streptomyces griseus
protease B, a member of the chymotrypsin superfamily, is encoded by a gene that expresses a pre-pro-mature protein. During secretion the precursor protein is processed into a mature, fully folded protease. In this study, we constructed a family of genes which encode deletions at the amino-terminal end of the propeptide. The secretion of active protease B was seen to decrease in an exponential manner according to the length of the deletion. The results underscore the intimate relationship between folding and secretion in bacterial protease expression. They further suggest that the propeptide segment of the zymogen stabilizes the folding of the mature enzyme through many small binding interactions over the entire surface of the peptide rather than through a few specific contacts.
Characterization of the
glnK-amtB
Operon of
Azotobacter vinelandii
Authors: Meletzus Dietmar , Rudnick Paul , Doetsch Natalie , Green Andrew , Kennedy Christina
Published: December 2019

To determine whether in
Azotobacter vinelandii
the P
II
protein influences the regulation of
nif
gene expression in response to fluxes in the ammonium supply, the gene encoding P
II
was isolated and characterized. Its deduced translation product was highly similar to P
II
proteins from other organisms, with the greatest degree of relatedness being exhibited to the
Escherichia coli glnK
gene product. A gene designated
amtB
was found downstream of and was cotranscribed with
glnK
as in
E. coli
. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote.
glnK
and
amtB
comprise an operon. Attempts to isolate a stable
glnK
mutant strain were unsuccessful, suggesting that
glnK
, like
glnA
, is an essential gene in
A. vinelandii
.
amtB
mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [
14
C]methylammonium.
Efflux Pumps Involved in Toluene Tolerance in
Pseudomonas putida
DOT-T1E
Authors: Ramos Juan L. , Duque Estrella , Godoy Patricia , Segura Ana
Published: December 2019

The basic mechanisms underlying solvent tolerance in
Pseudomonas putida
DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant
P. putida
DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of
P. putida
DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to
P. putida
DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn
5
-′
phoA
mutagenesis of wild-type
P. putida
DOT-T1E cells. The mutant, called
P. putida
DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[
14
C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[
14
C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of
14
C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in
P. putida
DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene
mexB
, which belongs to the efflux pump family of the resistant nodulator division type.
Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various
Bacillus
Species
Authors: Griffiths A. A. , Andersen P. A. , Wake R. G.
Published: December 2019

The replication terminator protein (RTP) of
Bacillus subtilis
interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in
Escherichia coli
, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to
B. subtilis
(
B. atrophaeus
,
B. amyloliquefaciens
,
B. mojavensis
, and
B. vallismortis
) have been identified and characterized. An RTP homolog could not be identified in another closely related species,
B. licheniformis
. The nucleotide and amino acid changes from
B. subtilis
among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the
rtp
genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from
B. licheniformis
. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the
B. mojavensis
RTP corresponds exactly to that purposefully introduced by others into
B. subtilis
RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in
B. subtilis
in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing the definition of a
Bacillus
terminator consensus and identification of two more terminators in the
B. subtilis
168 genome sequence to bring the total to nine.
Membrane-Bound Lytic Endotransglycosylase in
Escherichia coli
Authors: Kraft Angelika R. , Templin Markus F. , Höltje Joachim-Volker
Published: December 2019

The gene for a novel endotype membrane-bound lytic transglycosylase,
emtA
, was mapped at 26.7 min of the
E. coli
chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22 kDa. Overexpression of the
emtA
gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.
Mutants of
Citrobacter freundii
That Transport and Utilize Melibiose
Authors: Okazaki Noriko , Xu Xing Jue , Shimamoto Toshi , Kuroda Masayuki , Wilson Thomas H. , Tsuchiya Tomofusa
Published: December 2019

We have isolated mutants of
Citrobacter freundii
that can grow on melibiose. Inducible α-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized with
melB
(the gene for the melibiose transporter) DNA of
Escherichia coli
in the chromosomal DNA of wild-type
C. freundii
. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-β-
d
-thiogalactoside) transport in the mutant cells.
KtrAB, a New Type of Bacterial K
+
-Uptake System from
Vibrio alginolyticus
Authors: Nakamura Tatsunosuke , Yuda Rieko , Unemoto Tsutomu , Bakker Evert P.
Published: December 2019

Vibrio alginolyticus
contained two adjacent genes,
ktrA
and
ktrB
, which encode a new type of bacterial K
+
-uptake system. KtrA and KtrB are peripheral and integral membrane proteins, respectively. Six of the nine sequenced bacterial genomes contain homologs to both
ktrA
and
ktrB
, suggesting that KtrAB is widespread.
Identification of
cpxR
as a Positive Regulator Essential for Expression of the
Shigella sonnei virF
Gene
Authors: Nakayama Shu-ichi , Watanabe Haruo
Published: December 2019

virF
is the master regulator which activates the virulence determinant genes of
Shigella
spp. such as
ipaBCD
and
virG
. We previously reported that expression of
virF
itself is regulated in a pH-dependent manner and that
cpxA
, a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of
cpxR
, which has been thought to be the cognate response regulator of
cpxA
(J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolished
virF
expression almost completely. Purified CpxR bound directly to the upstream region of
virF
. Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate
virF
transcription in vitro. These results clearly indicated that CpxR was an essential activator for
virF
expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the
virF
gene induced a direct activation of
virF
transcription.
Regulation of Expression of
GLT1
, the Gene Encoding Glutamate Synthase in
Saccharomyces cerevisiae
Authors: Valenzuela Lourdes , Ballario Paola , Aranda Cristina , Filetici Patrizia , González Alicia
Published: December 2019

Saccharomyces cerevisiae
glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by
GLT1
. In this work, we analyzed
GLT1
transcriptional regulation.
GLT1-lacZ
fusions were prepared and
GLT1
expression was determined in a
GDH1
wild-type strain and in a
gdh1
mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in
GCN4
,
GLN3
,
GAT1/NIL1
, or
UGA43/DAL80
were transformed with a
GLT1-lacZ
fusion to determine whether the above-mentioned transcriptional factors had a role in
GLT1
expression. A collection of increasingly larger 5′ deletion derivatives of the
GLT1
promoter was constructed to identify DNA sequences that could be involved in
GLT1
transcriptional regulation. The effect of the lack of
GCN4
,
GLN3
, or
GAT1/NIL1
was also tested in the pertinent 5′ deletion derivatives. Our results indicate that (i)
GLT1
expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two
cis
-acting elements, a CGGN
15
CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in
GLT1
transcriptional activation; and (iii)
GLT1
expression is moderately regulated by
GCN4
under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
Expression of a Gene for a Porin-Like Protein of the OmpA Family fromMycobacterium tuberculosisH37Rv
Authors: Senaratne Ryan H. , Mobasheri Hamid , Papavinasasundaram K. G. , Jenner Peter , Lea Edward J. A. , Draper Philip
Published: December 2019

An open reading frame in the genomic database ofMycobacterium tuberculosisH37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned intoEscherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of brokenM. tuberculosiscells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed inM. tuberculosiscells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule fromM. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.