bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli tol-pal
Mutants Form Outer Membrane Vesicles
Authors: Bernadac Alain , Gavioli Marthe , Lazzaroni Jean-Claude , Raina Satish , Lloubès Roland
Published: December 2019

Mutations in the
tol-pal
genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as
tolC
,
lpp
, and
rfa
mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the
tol-pal
genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in
tolC
and
rfaD
cells in the same conditions. A
tolA
deletion in three different
Escherichia coli
strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of
tol
strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.
Molecular Analysis of the Capsule Gene Region of Group A
Streptococcus
: the
hasAB
Genes Are Sufficient for Capsule Expression
Authors: Ashbaugh Cameron D. , Albertí Sebastián , Wessels Michael R.
Published: December 2019

Enzymes directing the biosynthesis of the group A streptococcal hyaluronic acid capsule are encoded in the
hasABC
gene cluster. Inactivation of
hasC
, encoding UDP-glucose pyrophosphorylase in the heavily encapsulated group A streptococcal strain 87-282, had no effect on capsule production, indicating that
hasC
is not required for hyaluronic acid synthesis and that an alternative source of UDP-glucose is available for capsule production. Nucleotide sequence and deletion mutation analysis of the 5.5 kb of DNA upstream of
hasA
revealed that this region is not required for capsule expression. Many (10 of 23) group A streptococcal strains were found to contain insertion element IS
1239
′ approximately 50 nucleotides upstream of the −35 site of the
hasA
promoter. The presence of IS
1239
′ upstream of
hasA
did not prevent capsule expression. These results elucidate the molecular architecture of the group A streptococcal chromosomal region upstream of the
has
operon, indicate that
hasABC
are the sole components of the capsule gene cluster, and demonstrate that
hasAB
are sufficient to direct capsule synthesis in group A streptococci.
Isolation of
Candida glabrata
Homologs of the
Saccharomyces cerevisiae KRE9
and
KNH1
Genes and Their Involvement in Cell Wall β-1,6-Glucan Synthesis
Authors: Nagahashi Shigehisa , Lussier Marc , Bussey Howard
Published: December 2019

The
Candida glabrata KRE9
(
CgKRE9
) and
KNH1
(
CgKNH1
) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a
Saccharomyces cerevisiae
mutant with the disrupted
KNH1
locus and the
KRE9
gene placed under the control of a tetracycline-responsive promoter. Although a
cgknh1Δ
mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of
CgKRE9
resulted in several phenotypes similar to those of the
S. cerevisiae kre9Δ
null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of β-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in
C. glabrata
of the cognate
CgKRE9
promoter with the tetracycline-responsive promoter in a
cgknh1Δ
background rendered cell growth tetracycline sensitive on media containing glucose or galactose.
cgkre9Δ
cells were shown to be sensitive to calcofluor white specifically on glucose medium. In
cgkre9
mutants grown on glucose medium, cellular chitin levels were massively increased.
Sucrose Is a Nonaccumulated Osmoprotectant in
Sinorhizobium meliloti
Authors: Gouffi Kamila , Pichereau Vianney , Rolland Jean-Paul , Thomas Daniel , Bernard Théophile , Blanco Carlos
Published: December 2019

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of
Sinorhizobium meliloti
cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and
N
-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for
S. meliloti
. Overall, osmoregulation in
S. meliloti
appears to be strongly divergent from that in most bacteria.
Effect of Slow Growth on Metabolism of
Escherichia coli
, as Revealed by Global Metabolite Pool (“Metabolome”) Analysis
Authors: Tweeddale Helen , Notley-McRobb Lucinda , Ferenci Thomas
Published: December 2019

Escherichia coli
growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions. The extent of pool changes was followed through two-dimensional thin-layer chromatography of all
14
C-glucose labelled compounds extracted from bacteria. The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions. Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates. The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly. The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis. Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate. As RpoS controls a number of metabolic genes in response to nutrient limitation, an
rpoS
mutant was also analyzed for metabolite pools. The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS. These results indicate that total metabolite pool (“metabolome”) analysis offers a means of revealing novel aspects of cellular metabolism and global regulation.
CheZ Has No Effect on Flagellar Motors Activated by CheY
13DK106YW
Authors: Scharf Birgit E. , Fahrner Karen A. , Berg Howard C.
Published: December 2019

The behaviors of both
cheZ
-deleted and wild-type cells of
Escherichia coli
were found to be very sensitive to the level of expression of CheZ, a protein known to accelerate the dephosphorylation of the response regulator CheY-phosphate (CheY-P). However, cells induced to run and tumble by the unphosphorylated mutant protein CheY
13DK106YW
(CheY**) failed to respond to CheZ, even when CheZ was expressed at high levels. Therefore, CheZ neither affects the flagellar motors directly nor sequesters CheY**. In in vitro cross-linking studies, CheY** promoted trimerization of CheZ to the same extent as wild-type CheY but failed to induce the formation of complexes of higher molecular weight observed with CheY-P. Also, CheY** could be cross-linked to FliM, the motor receptor protein, nearly as well as CheY-P. Thus, to CheZ, CheY** looks like CheY, but to FliM, it looks like CheY-P.
hrcA
, Encoding the Repressor of the
groEL
Genes in
Streptomyces albus G
, Is Associated with a Second
dnaJ
Gene
Authors: Grandvalet Cosette , Rapoport Georges , Mazodier Philippe
Published: December 2019

Expression of the principal chaperones of the heat shock stimulon of
Streptomyces albus
G are under the negative control of different repressors. The
dnaK
operon is regulated by
hspR
, the last gene of the operon (
dnaK-grpE-dnaJ-hspR
).
hsp18
, encoding a member of the small heat shock protein family, is regulated by
orfY
, which is in the opposite orientation upstream of
hsp18
. The
groES-groEL1
operon and the
groEL2
gene are regulated differently. They present tandem copies of the CIRCE element found in the 5′ region of many heat shock genes and shown to act in
Bacillus subtilis
as an operator for a repressor encoded by
hrcA
(
hrc
stands for heat regulation at CIRCE). We report the identification in
S. albus
of a new heat shock operon containing
hrcA
and
dnaJ
homologs. Disruption of
hrcA
increased the transcription of the
groES-groEL1
operon and of the
groEL2
gene. These features were lost when the mutant was complemented in
trans
by an intact copy of
hrcA
. Despite considerable accumulation of the GroE chaperones in the
hrcA
mutant, there was no effect on formation of the aerial mycelium and sporulation, indicating that neither
hrcA
nor the level of
groE
gene expression is directly involved in the regulation of
Streptomyces
morphological differentiation.
The Gene for 16S rRNA Methyltransferase (
ksgA
) Functions as a Multicopy Suppressor for a Cold-Sensitive Mutant of Era, an Essential RAS-Like GTP-Binding Protein in
Escherichia coli
Authors: Lu Qing , Inouye Masayori
Published: December 2019

Era, a Ras-like GTP-binding protein in
Escherichia coli
, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an
E. coli
genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the
E. coli
genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the
ksgA
gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.
Two Amino Acid Residues of Transposase Contributing to Differential Transposability of IS
1
Elements in
Escherichia coli
Authors: Chen Jiann-Hwa , Hsu Wen-Ben , Hwang Jiing-Luen
Published: December 2019

Escherichia coli
W3110 contains four types of IS
1
elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS
1
plasmid insertion mutants, which resulted from a minimum of 26 independent IS
1
insertion events. All of them had insertions of IS
1
of the IS
1
A (IS
1
E and IS
1
G) type. Inspection of the transposase sequences of the four IS
1
types and the IS
1
of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS
1
elements in W3110. The two amino acid residues of the transposase in IS
1
A (IS
1
E and IS
1
G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS
1
A (IS
1
E and IS
1
G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS
1
elements in W3110 was confirmed.
Catabolite Gene Activator Protein Mutations Affecting Activity of the
araBAD
Promoter
Authors: Zhang Xin , Schleif Robert
Published: December 2019

We have studied catabolite gene activator protein (CAP) activation at the
araBAD
promoter,

p
BAD

, in the absence of DNA looping. We ruled out the two most plausible indirect activation mechanisms: CAP-induced folding of upstream DNA back upon RNA polymerase, and CAP-induced stabilization of AraC binding to DNA. Therefore, a direct CAP-RNA polymerase interaction seemed likely. We sought and found CAP mutants defective in transcription activation at

p
BAD

that retained normal DNA binding affinity. Some mutations altered residues in the interval from positions 150 to 164 that includes CAP activating region 1 (AR1), which has been shown to contact RNA polymerase at a number of promoters. Unexpectedly, additional mutations were found that altered residues in the region between positions 46 and 68 and at position 133. This includes the region known as activating region 3 (AR3). Mutations from both groups also affect the
araFGH
and
rhaBAD
promoters.
A DnaK Homolog in
Myxococcus xanthus
Is Involved in Social Motility and Fruiting Body Formation
Authors: Yang Zhaomin , Geng Yongzhi , Shi Wenyuan
Published: December 2019

Myxococcus xanthus
is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of
M. xanthus
were isolated through Tn
5
transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the
dnaK
genes in other bacteria, this
M. xanthus
homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of
M. xanthus.
Growth-Independent Regulation of
CLN3
mRNA Levels by Nutrients in
Saccharomyces cerevisiae
Authors: Parviz Fereshteh , Heideman Warren
Published: December 2019

Saccharomyces cerevisiae
cells regulate progress through the G
1
phase of the cell cycle in response to nutrients, moving quickly through G
1
in rich medium and slowly in poor medium. Recent work has shown that the levels of Cln3 protein, a G
1
cyclin, are low in cells growing in poor medium and high in cells growing rapidly in rich medium, consistent with the previously recognized role of Cln3 in promoting passage through Start. Cln3 protein levels appear to be regulated both transcriptionally and posttranscriptionally. We have worked to define the nutrient signals that regulate
CLN3
mRNA levels. We find that
CLN3
mRNA levels are high during log-phase growth in glucose medium, low in postdiauxic cells growing on ethanol, and slightly lower still in cells in stationary phase.
CLN3
mRNA levels are induced by glucose in a process that involves transcriptional control, requires metabolism of the glucose, and is independent of the Ras-cyclic AMP pathway.
CLN3
mRNA levels are also positively regulated by nitrogen sources, but phosphorus and sulfur limitation do not affect
CLN3
message levels.
Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes
kpsD
,
kpsM
,
kpsT
, and
kpsE
from an Extraintestinal Isolate of
Escherichia coli
(CP9, O4/K54/H5)
Authors: Russo Thomas A. , Wenderoth Suzanne , Carlino Ulrike B. , Merrick Joseph M. , Lesse Alan J.
Published: December 2019

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of
Escherichia coli
, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, Tn
phoA
-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes
kpsDMTE
. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although
kps
K54
DMTE
are significantly divergent from their group II homologs at both the DNA and protein levels
phoA
fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps
K54
M and Kps
K54
T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps
K54
E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated
kpsM
(
kpsM
truncated
) and its 5′ noncoding regulatory sequence were identified. In contrast to the complete
kps
K54
M
, this region was highly homologous to the group II
kpsM
. Fifty-three base pairs 3′ from the end of
kpsM
truncated
was a sequence 75% homologous to the 39-bp inverted repeat in the IS
110
insertion element from
Streptomyces coelicolor
. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS
110
-mediated horizontal transfer.
Construction and Characterization of a
Helicobacter pylori clpB
Mutant and Role of the Gene in the Stress Response
Authors: Allan Elaine , Mullany Peter , Tabaqchali Soad
Published: December 2019

Antiserum raised against whole
Helicobacter pylori
cells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic
H. pylori clpB
mutant showed increased sensitivity to high-temperature stress, indicating that the
clpB
gene product functions as a stress response protein in
H. pylori.
Characterization of Multiple Regions Involved in Replication and Mobilization of Plasmid pNZ4000 Coding for Exopolysaccharide Production in
Lactococcus lactis
Authors: van Kranenburg Richard , de Vos Willem M.
Published: December 2019

We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in
Lactococcus lactis
NIZO B40. The plasmid contains four highly conserved replication regions with homologous
rep
genes (
repB1
,
repB2
,
repB3
, and
repB4
) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in
L. lactis
. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different
L. lactis
strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an
oriT
sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these
oriT
sequences, demonstrating their functionality. One
oriT
sequence was followed by a
mobA
gene, coding for a
trans
-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and the
oriT
sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and
nic
site of the
oriT
from the
Escherichia coli
plasmid R64. The presence on pNZ4000 of four functional replicons, two
oriT
sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.
Conserved Structural Regions Involved in the Catalytic Mechanism of
Escherichia coli
K-12 WaaO (RfaI)
Authors: Shibayama Keigo , Ohsuka Shinji , Tanaka Toshihiko , Arakawa Yoshichika , Ohta Michio
Published: December 2019

Escherichia coli
K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of
E. coli
K-12 WaaO revealed four critical amino acid residues.
Regulation of the
Bacillus subtilis
GlcT Antiterminator Protein by Components of the Phosphotransferase System
Authors: Bachem Steffi , Stülke Jörg
Published: December 2019

Bacillus subtilis
utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the
ptsGHI
operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EII
Glc
), the product of
ptsG
. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EII
Glc
. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with
B. subtilis
glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.
Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein of
Agrobacterium tumefaciens
Authors: Zhu Jun , Beaber John W. , Moré Margret I. , Fuqua Clay , Eberhard Anatol , Winans Stephen C.
Published: December 2019

The TraR and TraI proteins of
Agrobacterium tumefaciens
mediate cell-density-dependent expression of the Ti plasmid
tra
regulon. TraI synthesizes the autoinducer pheromone
N
-(3-oxooctanoyl)-
l
-homoserine lactone (3-oxo-C
8
-HSL), while TraR is an 3-oxo-C
8
-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C
8
-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C
8
-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type
A. tumefaciens
released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C
8
-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with
tra
gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C
8
-HSL significantly stimulates
tra
gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.
Methanococcus jannaschii
Flap Endonuclease: Expression, Purification, and Substrate Requirements
Authors: Rao H. G. V. , Rosenfeld Amy , Wetmur James G.
Published: December 2019

The flap endonuclease (FEN) of the hyperthermophilic archaeon
Methanococcus jannaschii
was expressed in
Escherichia coli
and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of
Taq
DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for
Taq
DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion φX174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.
Fumarate Regulation of Gene Expression in
Escherichia coli
by the DcuSR (
dcuSR
Genes) Two-Component Regulatory System
Authors: Zientz Evelyn , Bongaerts Johannes , Unden Gottfried
Published: December 2019

In
Escherichia coli
the genes encoding the anaerobic fumarate respiratory system are transcriptionally regulated by C
4
-dicarboxylates. The regulation is effected by a two-component regulatory system, DcuSR, consisting of a sensory histidine kinase (DcuS) and a response regulator (DcuR). DcuS and DcuR are encoded by the
dcuSR
genes (previously
yjdHG
) at 93.7 min on the calculated
E. coli
map. Inactivation of the
dcuR
and
dcuS
genes caused the loss of C
4
-dicarboxylate-stimulated synthesis of fumarate reductase (
frdABCD
genes) and of the anaerobic fumarate-succinate antiporter DcuB (
dcuB
gene). DcuS is predicted to contain a large periplasmic domain as the supposed site for C
4
-dicarboxylate sensing. Regulation by DcuR and DcuS responded to the presence of the C
4
-dicarboxylates fumarate, succinate, malate, aspartate, tartrate, and maleate. Since maleate is not taken up by the bacteria under these conditions, the carboxylates presumably act from without. Genes of the aerobic C
4
-dicarboxylate pathway encoding succinate dehydrogenase (
sdhCDAB
) and the aerobic succinate carrier (
dctA
) are only marginally or negatively regulated by the DcuSR system. The CitAB two-component regulatory system, which is highly similar to DcuSR, had no effect on C
4
-dicarboxylate regulation of any of the genes.
Different Phenotypic Classes of
Sinorhizobium meliloti
Mutants Defective in Synthesis of K Antigen
Authors: Campbell Gordon R. O. , Reuhs Bradley L. , Walker Graham C.
Published: December 2019

For
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
) AK631 to establish effective symbiosis with alfalfa, it must be able to synthesize a symbiotically active form of its K antigen, a capsular polysaccharide containing a Kdo (3-deoxy-
d
-manno-octulosonic acid) derivative. Previously isolated mutants defective in the synthesis of K antigen are resistant to bacteriophage φ16-3. By screening ca. 100,000 Tn
5
-mutagenized
R. meliloti
bacteria for resistance to bacteriophage φ16-3, we isolated 119 mutants, 31 of which could not be complemented by genes previously identified as being required for K-antigen synthesis. Of these 31 new mutants, 13 were symbiotically defective and lacked the K antigen. Through genetic and phenotypic analyses, we have grouped these mutants into four distinct classes. Although all of these mutants lack the K antigen, many also have altered lipopolysaccharides (LPS), suggesting that the biochemical pathways for the synthesis of K antigen and LPS have common enzymatic steps. In addition, we have found that these and other classes of K-antigen-defective mutants of
S. meliloti
AK631 exhibit unique patterns of sensitivities to phage strains to which the parental strain was resistant. Our studies have identified new classes of genes required for both the synthesis of K antigen and the symbiotic proficiency of
S. meliloti
AK631. Some of these classes of genes also play a role in LPS synthesis.
Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product
Authors: Beller Harry R. , Spormann Alfred M.
Published: December 2019

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(
R
)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.
Oxygen-Insensitive Nitroreductases: Analysis of the Roles of
nfsA
and
nfsB
in Development of Resistance to 5-Nitrofuran Derivatives in
Escherichia coli
Authors: Whiteway J. , Koziarz P. , Veall J. , Sandhu N. , Kumar P. , Hoecher B. , Lambert I. B.
Published: December 2019

Nitroheterocyclic and nitroaromatic compounds constitute an enormous range of chemicals whose potent biological activity has significant human health and environmental implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the
nfsA
and
nfsB
genes of a large number of nitrofuran-resistant mutants of
Escherichia coli
and have correlated mutation with cell extract nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an
nfsA
mutation, while the increased resistance associated with second-step mutants is a consequence of an
nfsB
mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, especially with regard to the identification of flavin mononucleotide binding sites. We show that expression of plasmid-carried
nfsA
and
nfsB
genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and 49%, respectively, contained insertion sequence elements in
nfsA
and
nfsB
. IS
1
integrated in both genes, while IS
30
and IS
186
were found only in
nfsA
and IS
2
and IS
5
were observed only in
nfsB
. Insertion hot spots for IS
30
and IS
186
are indicated in
nfsA
, and a hot spot for IS
5
insertion is evident in
nfsB
. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in
nfsA
and
nfsB
.
Characterization of Circular Plasmid Dimers in
Borrelia burgdorferi
Authors: Tilly Kit , Lubke Lori , Rosa Patricia
Published: December 2019

We have inactivated the
ospC
,
oppAIV
, and
guaB
genes on the 26-kb circular plasmid of
Borrelia burgdorferi
(cp26) by allelic exchange. On several occasions following such transformations, the cp26 of transformants had an aberrant mobility through agarose gels. Characterization of these cp26 molecules showed that the plasmid had dimerized. These dimers were quite stable during either selective or nonselective passage. Subsequent transformations with dimer DNA supported the hypothesis that in
B. burgdorferi
, transforming cp26 DNA most likely does not displace the resident homologous plasmid but rather must recombine in order to donate sequences that it carries. These serendipitous findings provide a mechanism for obtaining heterozygous complemented control strains when mutant phenotypes are characterized.
A Cytochrome
cbb
3
(Cytochrome c) Terminal Oxidase in
Azospirillum brasilense
Sp7 Supports Microaerobic Growth
Authors: Marchal Kathleen , Sun Jun , Keijers Veerle , Haaker Huub , Vanderleyden Jos
Published: December 2019

Spectral analysis indicated the presence of a cytochrome
cbb
3
oxidase under microaerobic conditions in
Azospirillum brasilense
Sp7 cells. The corresponding genes (
cytNOQP
) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an
A. brasilense cytN
mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (μ
e
) of the
A. brasilense cytN
mutant was comparable to the wild-type specific growth rate (μ
e
of approximately 0.2 h
−1
). In microaerobic NH
4
+
-supplemented conditions, the low respiration of the
A. brasilense cytN
mutant affected its specific growth rate (μ
e
of approximately 0.02 h
−1
) compared to the wild-type specific growth rate (μ
e
of approximately 0.2 h
−1
). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH
4
+
-supplemented conditions. Differences in growth rates and respiration between the wild type and the
A. brasilense cytN
mutant were less pronounced under these nitrogen-fixing conditions (μ
e
of approximately 0.03 h
−1
for the wild type and 0.02 h
−1
for the
A. brasilense cytN
mutant). The nitrogen-fixing capacity of the
A. brasilense cytN
mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the
A. brasilense
cytochrome
cbb
3
oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.
α-Galactoside Uptake in
Rhizobium meliloti
: Isolation and Characterization of
agpA
, a Gene Encoding a Periplasmic Binding Protein Required for Melibiose and Raffinose Utilization
Authors: Gage Daniel J. , Long Sharon R.
Published: December 2019

Rhizobium meliloti
can occupy at least two distinct ecological niches; it is found in the soil as a free-living saprophyte, and it also lives as a nitrogen-fixing intracellular symbiont in root nodules of alfalfa and related legumes. One approach to understanding how
R. meliloti
alters its physiology in order to become an integral part of a developing nodule is to identify and characterize genes that are differentially expressed by bacteria living inside nodules. We used a screen to identify genes under the control of the
R. meliloti
regulatory protein NodD3, SyrM, or SyrA. These regulatory proteins are expressed by bacteria growing inside the root nodule. One gene isolated in this screen was mapped to pSymB and displayed complex regulation. The gene was downregulated by the
syrA
gene product and also by glucose and succinate. This gene, referred to as
agpA
, encodes a periplasmic binding protein that is most similar to proteins from the periplasmic oligopeptide binding protein family. It is likely that AgpA binds α-galactosides, because α-galactosides induce the expression of
agpA
, and
agpA
mutants cannot utilize or transport these sugars. Activity of an
agpA
::Tn
phoA
fusion was downregulated by SyrA. Because
syrA
is known to be expressed at high levels in intracellular symbiotic
R. meliloti
and at low levels in the free-living bacteria, we propose that AgpA may belong to the class of gene products whose expression decreases when
R. meliloti
becomes an intracellular symbiont.
Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide inStreptococcus mutans
Authors: Yamashita Yoshihisa , Tsukioka Yuichi , Tomihisa Kiyotaka , Nakano Yoshio , Koga Toshihiko
Published: December 2019

We identified inStreptococcus mutanssix new genes (rgpAthroughrgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in thergpEmutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.
Regulation and Physiological Role of the
DAS1
Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast
Candida boidinii
Authors: Sakai Yasuyoshi , Nakagawa Tomoyuki , Shimase Masayuki , Kato Nobuo
Published: December 2019

The physiological role of dihydroxyacetone synthase (DHAS) in
Candida boidinii
was evaluated at the molecular level. The
DAS1
gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that
DAS1
expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The
DAS1
gene was disrupted in one step in the host genome (
das1
Δ strain), and the growth of the
das1
Δ strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The
das1
Δ strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480–4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the
das1
Δ strain on media containing methylamine or choline as a nitrogen source) suggested that
DAS1
is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.
In Vivo Assay of Protein-Protein Interactions in Hin-Mediated DNA Inversion
Authors: Lee Sun Young , Lee Hee Jung , Lee Heejin , Kim Shukho , Cho Eun Hee , Lim Heon Man
Published: December 2019

In order to form the catalytic nucleoprotein complex called the invertasome in the Hin-mediated DNA inversion reaction, interactions of the DNA-binding proteins Hin and Fis are required. Assays for these protein-protein interactions have been exploited with protein cross-linkers in vitro. In this study, an in vivo assay system that probes protein-protein interactions was developed. The formation of a DNA loop generated by protein interactions resulted in transcriptional repression of an artificially designed operon, which in turn increased the chance of survival of
Escherichia coli
host cells in a streptomycin-containing medium. Using this system, we were able to assay the Hin-Hin interaction that results in the pairing of the two recombination sites and protein interactions that result in the formation of the invertasome. This assay system also led us to find that an individual Hin dimer bound on a recombination site can form a stable complex with Fis bound on the recombinational enhancer; this finding has never been observed in in vitro studies. Possible pathways toward the formation of the invertasome are discussed based on the assay results for a previously reported Hin mutant.
The
yiaE
Gene, Located at 80.1 Minutes on the
Escherichia coli
Chromosome, Encodes a 2-Ketoaldonate Reductase
Authors: Yum Do-Young , Lee Bong-Yong , Hahm Dae-Hyum , Pan Jae-Gu
Published: December 2019

An open reading frame located in the
bisC-cspA
intergenic region, or at 80.1 min on the
Escherichia coli
chromosome, encodes a hypothetical 2-hydroxyacid dehydrogenase, which was identified as a result of the
E. coli
Genome Sequencing Project. We report here that the product of the gene (
yiaE
) is a 2-ketoaldonate reductase (2KR). The gene was cloned and expressed with a C-terminal His tag in
E. coli
, and the protein was purified by metal-chelate affinity chromatography. The determination of the NH
2
-terminal amino acid sequence of the protein defined the translational start site of this gene. The enzyme was found to be a 2KR catalyzing the reduction of 2,5-diketo-
d
-gluconate to 5-keto-
d
-gluconate, 2-keto-
d
-gluconate (2KDG) to
d
-gluconate, 2-keto-
l
-gulonate to
l
-idonate. The reductase was optimally active at pH 7.5, with NADPH as a preferred electron donor. The deduced amino acid sequence showed 69.4% identity with that of 2KR from
Erwinia herbicola
. Disruption of this gene on the chromosome resulted in the loss of 2KR activity in
E. coli. E. coli
W3110 was found to grow on 2KDG, whereas the mutant deficient in 2KR activity was unable to grow on 2KDG as the carbon source, suggesting that 2KR is responsible for the catabolism of 2KDG in
E. coli
and the diminishment of produced 2KDG from
d
-gluconate in the cultivation of
E. coli
harboring a cloned gluconate dehydrogenase gene.
Anaerobic Expression ofEscherichia coliSuccinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth
Authors: Maklashina Elena , Berthold Deborah A. , Cecchini Gary
Published: December 2019

Succinate-ubiquinone oxidoreductase (SQR) fromEscherichia coliis expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration byE. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of thefrdABCDoperon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth ofE. colibecause expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the PFRDpromoter of thefrdABCDoperon fused to thesdhCDABgenes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth ofE. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex,E. coliaccommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.
Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers
Authors: Higgs Penelope I. , Myers Paul S. , Postle Kathleen
Published: December 2019

The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.
The
Escherichia coli cmlA
Gene Encodes the Multidrug Efflux Pump Cmr/MdfA and Is Responsible for Isopropyl-β-
d
-Thiogalactopyranoside Exclusion and Spectinomycin Sensitivity
Authors: Bohn Chantal , Bouloc Philippe
Published: December 2019

Expression of cloned genes from isopropyl-β-
d
-thiogalactopyranoside (IPTG)-regulated promoters is lowered when the
Escherichia coli
CmlA/Cmr/MdfA efflux pump is overexpressed, probably due to IPTG exclusion from the cytoplasm. The previously reported
cmlA1
mutation confers a similar phenotype.
cmlA1
contains an IS
30
insertion upstream of
cmr/mdfA
, which creates a putative promoter. CmlA overproduction also causes spectinomycin hypersensitivity.