bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Two-Component Signal Transduction System from
Corynebacterium diphtheriae
That Activates Gene Expression in Response to the Presence of Heme and Hemoglobin
Authors: Schmitt Michael P.
Published: December 2019

Corynebacterium diphtheriae
, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The
C. diphtheriae hmuO
gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the
hmuO
gene in
C. diphtheriae
is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a
C. diphtheriae
chromosomal library were shown to activate transcription from the
hmuO
promoter in
Escherichia coli
. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the
C. diphtheriae
chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (
chrA
) and its cognate sensor kinase (
chrS
), was shown to strongly activate transcription from the
hmuO
promoter in a heme-dependent manner in
E. coli
. A mutation in
chrA
(
chrAD50N
), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the
hmuO
promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.
Cold Shock Response of
Bacillus subtilis
: Isoleucine-Dependent Switch in the Fatty Acid Branching Pattern for Membrane Adaptation to Low Temperatures
Authors: Klein Wolfgang , Weber Michael H. W. , Marahiel Mohamed A.
Published: December 2019

Bacillus subtilis
has developed sophisticated mechanisms to withstand fluctuations in temperature. Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane’s function at all temperatures. The fatty acid profile of
B. subtilis
is characterized by a high content of branched fatty acids irrespective of the growth medium. Here, we report on the importance of isoleucine for
B. subtilis
to survive cold shock from 37 to 15°C. Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis. Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective. Fatty acid profiles of different
B. subtilis
wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock. There were no significant changes in the fatty acid saturation or acyl chain length. The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile. The switch to a fatty acid profile dominated by anteiso-C
15:0
and C
17:0
at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of
B. subtilis
in the cold.
Regulation of Cell Component Production by Growth Rate in the Group BStreptococcus
Authors: Ross Robin A. , Madoff Lawrence C. , Paoletti Lawrence C.
Published: December 2019

Group BStreptococcus(GBS) is the leading cause of bacterial sepsis and meningitis among neonates. While the capsular polysaccharide (CPS) is an important virulence factor of GBS, other cell surface components, such as C proteins, may also play a role in GBS disease. CPS production by GBS type III strain M781 was greater when cells were held at a fast (1.4-h mass-doubling time [td]) than at a slow (11-htd) rate of growth. To further investigate growth rate regulation of CPS production and to investigate production of other cell components, different serotypes and strains of GBS were grown in continuous culture in a semidefined and a complex medium. Samples were obtained after at least five generations at the selected growth rate. Cells and cell-free supernatants were processed immediately, and results from all assays were normalized for cell dry weight. All serotypes (Ia, Ib, and III) and strains (one or two strains per serotype) tested produced at least 3.6-fold more CPS at atdof 1.4 h than at atdof 11 h. Production of beta C protein by GBS type Ia strain A909 and type Ib strain H36B was also shown to increase at least 5.5-fold with increased growth rate (production at atdof 1.4 h versus 11 h). The production of alpha C protein by the same strains did not significantly change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate regulation is specific for select cell components in GBS, including beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.
Two Different Types of Dehalogenases, LinA and LinB, Involved in γ-Hexachlorocyclohexane Degradation in
Sphingomonas paucimobilis
UT26 Are Localized in the Periplasmic Space without Molecular Processing
Authors: Nagata Yuji , Futamura Akiko , Miyauchi Keisuke , Takagi Masamichi
Published: December 2019

γ-Hexachlorocyclohexane (γ-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a γ-HCH-degrading bacterium,
Sphingomonas paucimobilis
UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (γ-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of γ-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.
Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from
Pseudomonas aeruginosa
Authors: Ochs Martina M. , Lu Chung-Dar , Hancock Robert E. W. , Abdelal Ahmed T.
Published: December 2019

Pseudomonas aeruginosa
can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of
oprD
, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the
oprD
gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of
oprD
is linked to both carbon and nitrogen metabolism of
Pseudomonas aeruginosa
.
Regulation of Synthesis of the
Bacillus subtilis
Transition-Phase, Spore-Associated Antibacterial Protein TasA
Authors: Stöver Axel G. , Driks Adam
Published: December 2019

Previously, we identified a novel component of
Bacillus subtilis
spores, called TasA, which possesses antibacterial activity. TasA is made early in spore formation, as cells enter stationary phase, and is secreted into the medium as well as deposited into the spore. Here, we show that
tasA
expression can occur as cells enter stationary phase even under sporulation-repressing conditions, indicating that TasA is a transition-phase protein.
tasA
and two upstream genes,
yqxM
and
sipW
, likely form an operon, transcription of which is under positive control by the transition-phase regulatory genes
spo0A
and
spo0H
and negative control by the transition phase regulatory gene
abrB
. These results are consistent with the suggestion that
yqxM
,
sipW
, and
tasA
constitute a transition phase operon that could play a protective role in a variety of cellular responses to stress during late-exponential-phase and early-stationary-phase growth in
B. subtilis
.
A Unique α-1,3 Mannosyltransferase of the Pathogenic Fungus
Cryptococcus neoformans
Authors: Doering Tamara L.
Published: December 2019

The major virulence factor of the pathogenic fungus
Cryptococcus neoformans
is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear α-1,3-linked mannan substituted with
d
-xylose and
d
-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an α-1,3-dimannoside acceptor, forming a second α-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast
Saccharomyces cerevisiae
, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from
C. neoformans
membranes adds mannose in α-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the α-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.
Characterization of
Pseudomonas aeruginosa
Enoyl-Acyl Carrier Protein Reductase (FabI): a Target for the Antimicrobial Triclosan and Its Role in Acylated Homoserine Lactone Synthesis
Authors: Hoang Tung T. , Schweizer Herbert P.
Published: December 2019

The
Pseudomonas aeruginosa fabI
structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that
fabI
is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal
fabI
gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The
fabI
-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the
fabI
mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly
95
-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified
P. aeruginosa N
-butyryl-
l
-homoserine lactone (C
4
-HSL) synthase, RhlI, could synthesize C
4
-HSL from crotonyl-ACP and
S
-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C
4
-HSL and
N
-(3-oxo)-dodecanoyl-
l
-homoserine lactones were reduced 50% in a
fabI
mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
Characterization of a
Pseudomonas aeruginosa
Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)
Authors: Kutchma Alecksandr J. , Hoang Tung T. , Schweizer Herbert P.
Published: December 2019

A DNA fragment containing the
Pseudomonas aeruginosa fabD
(encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase),
fabG
(encoding β-ketoacyl-ACP reductase),
acpP
(encoding ACP), and
fabF
(encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This
fab
gene cluster is delimited by the
plsX
(encoding a poorly understood enzyme of phospholipid metabolism) and
pabC
(encoding 4-amino-4-deoxychorismate lyase) genes; the
fabF
and
pabC
genes seem to be translationally coupled. The
fabH
gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between
plsX
and
fabD
, is absent from this gene cluster. A chromosomal temperature-sensitive
fabD
mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal
fabF
insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of
cis
-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal
fabG
gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H
6
-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption–ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4′-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H
6
-FabD exhibited malonyl-CoA:ACP transacylase activity.
Clustering of the Chemoreceptor Complex in
Escherichia coli
Is Independent of the Methyltransferase CheR and the Methylesterase CheB
Authors: Lybarger Suzanne R. , Maddock Janine R.
Published: December 2019

The
Escherichia coli
chemoreceptors and their associated cytoplasmic proteins, CheA and CheW, cluster predominantly at the cell poles. The nature of the clustering remains a mystery. Recent studies suggest that CheR binding to and/or methylation of the chemoreceptors may play a role in chemoreceptor complex aggregation. In this study, we examined the intracellular distribution of the chemoreceptors by immunoelectron microscopy in strains lacking either the methyltransferase CheR or the methylesterase CheB. The localization data revealed that, in vivo, aggregation of the chemoreceptor complex was independent of either CheR or CheB.
TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in
Escherichia coli
Authors: Schmidt-Eisenlohr Heike , Domke Natalie , Baron Christian
Published: December 2019

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related
Agrobacterium tumefaciens
T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of
Escherichia coli
allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,
traM
,
traB
,
traD
, and
traE
but not
eex
, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in
E. coli
carrying pKM101 but not derivatives with transposon insertions in essential
tra
genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in
A. tumefaciens
partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.
HP0333, a Member of the
dprA
Family, Is Involved in Natural Transformation in
Helicobacter pylori
Authors: Ando Takafumi , Israel Dawn A. , Kusugami Kazuo , Blaser Martin J.
Published: December 2019

Helicobacter pylori
is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For
Haemophilus influenzae
,
dprA
is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of
H. pylori
26695 revealed a
dprA
homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to
dprA
of
H. influenzae
, HP0333, present in each of 11 strains studied, was disrupted in two
H. pylori
isolates. For both mutants, the frequency of transformation by
H. pylori
chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in
H. influenzae
. Complementation of the mutant with HP0333 inserted in
trans
in the chromosomal
ureAB
locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while
dprA
is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.
Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in
Streptomyces cinnamonensis
: Influence on Polyketide Antibiotic Biosynthesis
Authors: Vrijbloed Jan W. , Zerbe-Burkhardt Katja , Ratnatilleke Ananda , Grubelnik-Leiser Andreas , Robinson John A.
Published: December 2019

The coenzyme B
12
-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of
n
-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of
n
- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer
Streptomyces cinnamonensis
. Mutants prepared by inserting a hygromycin resistance gene (
hygB
) into either
icmA
or
mutB
, encoding the large subunits of ICM and MCM, respectively, have been characterized. The
icmA
::
hygB
mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The
mutB
::
hygB
mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources.
13
C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence,
n
-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.
Genomic Subtraction Identifies
Salmonella typhimurium
Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon,
stf
, Which Are Absent in
Salmonella typhi
Authors: Emmerth Melanie , Goebel Werner , Miller Samuel I. , Hueck Christoph J.
Published: December 2019

Salmonella typhimurium
causes systemic and fatal infection in inbred mice, while the related serotype
Salmonella typhi
is avirulent for mammals other than humans. In order to identify genes from the virulent strain
S. typhimurium
ATCC 14028 that are absent in
S. typhi
Ty2, and therefore might be involved in
S. typhimurium
mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon,
stfACDEFG
, located at centisome 5 of the
S. typhimurium
chromosome, which is absent in
S. typhi
,
Salmonella arizonae
, and
Salmonella bongori
but was detected in several other
Salmonella
serotypes. The fimbrial genes represent a genomic insertion in
S. typhimurium
compared to the respective region between
fhuB
and
hemL
in
Escherichia coli
K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the
S. typhimurium
virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.
Changes in Cell Size and DNA Content in
Sulfolobus
Cultures during Dilution and Temperature Shift Experiments
Authors: Hjort Karin , Bernander Rolf
Published: December 2019

Stationary-phase cultures of different hyperthermophilic species of the archaeal genus
Sulfolobus
were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79°C were shifted to room temperature or to ice-water baths, the cells were found to “freeze” in mid-growth. After a shift back to 79°C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.
Identification of ς
B
-Dependent Genes in
Bacillus subtilis
Using a Promoter Consensus-Directed Search and Oligonucleotide Hybridization
Authors: Petersohn Anja , Bernhardt Jörg , Gerth Ulf , Höper Dirk , Koburger Torsten , Völker Uwe , Hecker Michael
Published: December 2019

A consensus-directed search for ς
B
promoters was used to locate potential candidates for new ς
B
-dependent genes in
Bacillus subtilis
. Screening of those candidates by oligonucleotide hybridizations with total RNA from exponentially growing or ethanol-stressed cells of the wild type as well as a
sigB
mutant revealed 22 genes that required ς
B
for induction by ethanol. Although almost 50% of the proteins encoded by the newly discovered ς
B
-dependent stress genes seem to be membrane localized, biochemical functions have so far not been defined for any of the gene products. Allocation of the genes to the ς
B
-dependent stress regulon may indicate a potential function in the establishment of a multiple stress resistance. AldY and YhdF show similarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestion that ς
B
-dependent proteins may be involved in the maintenance of the intracellular redox balance after stress.
Multiple Mechanisms Are Used for Growth Rate and Stringent Control of
leuV
Transcriptional Initiation in
Escherichia coli
Authors: Pokholok Dmitry K. , Redlak Maria , Turnbough Charles L. , Dylla Sara , Holmes Walter M.
Published: December 2019

Expression of the
Escherichia coli leuV
operon, which contains three tRNA
1
Leu
genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the
leuV
promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the
leuV
promoter occurs in the absence of guanosine 3′,5′-bisdiphosphate. To determine the mechanisms of the
leuV
promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the
leuV
promoter and its structural variants in vitro and in vivo. The
leuV
promoter open complexes were an order of magnitude more stable to heparin challenge than those of
rrnBp
1
. The major initiating nucleotide GTP as well as other NTPs increased the stability of the
leuV
promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in
leuV
promoter activity was seen. It therefore appears that transcription initiation from the
leuV
promoter is less sensitive to changes in intracellular NTP concentration than that from
rrnBp
1
. Comparative analysis of regulation of the
leuV
promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of
leuV
promoter mutations, which abolished GRDC of the
leuV
core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type
leuV
promoters containing UAS showed improved GRDC in a
fis
mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the
leuV
promoter.
Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase
Authors: Zinser Erik R. , Kolter Roberto
Published: December 2019

Starved cultures of
Escherichia coli
undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the
rpoS819
allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). Here we have identified three new GASP loci from an aged
rpoS819
strain:
sgaA
,
sgaB
, and
sgaC
. Each locus is capable of conferring GASP on the
rpoS819
parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.
Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of
Salmonella
Authors: Muramoto Kazumasa , Makishima Shigeru , Aizawa Shin-Ichi , Macnab Robert M.
Published: December 2019

The flagellar hook of
Salmonella
is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a
fliC
(flagellin) mutant, or a
fliD
(hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a
fliK
(hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a
flgK
(HAP1, first hook-filament junction protein) mutant or a
flgL
(HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.
Halophilic 20S Proteasomes of the Archaeon
Haloferax volcanii
: Purification, Characterization, and Gene Sequence Analysis
Authors: Wilson Heather L. , Aldrich Henry C. , Maupin-Furlow Julie
Published: December 2019

A 20S proteasome, composed of α
1
and β subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon
Haloferax volcanii
. The predominant peptide-hydrolyzing activity of the 600-kDa α
1
β-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75°C. The α
1
β-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the α
1
β-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active α
1
β-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the β-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and
N
-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the
H. volcanii
proteasome (

K
i

= 0.2 and 8 μM, respectively). In addition to the genes encoding the α
1
and β subunits, a gene encoding a second α-type proteasome protein (α
2
) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the α
1
β-proteasome resulted in the copurification of the α
2
protein with the α
1
and β subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the α
1
β-proteasome, suggesting that at least two separate 20S proteasomes are synthesized. This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different α subunits in this halophilic archaeon.
Study of the Interaction between Bacteriophage T4 asiA andEscherichia coliς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity toE. coliCells by Coexpression of a Truncated ς70Fragment
Authors: Sharma Umender K. , Ravishankar Sudha , Shandil Radha Krishan , Praveen P. V. K. , Balganesh T. S.
Published: December 2019

The interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coliς70was studied by using the yeast two-hybrid system. Truncation of ς70to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression inE. colicould be neutralized and the complex of truncated ς70and asiA could be expressed in large quantities and purified.
Synthetic Lethal Phenotypes Caused by Mutations Affecting Chromosome Partitioning in
Bacillus subtilis
Authors: Britton Robert A. , Grossman Alan D.
Published: December 2019

We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in
Bacillus subtilis
. Loss-of-function mutations in
spoIIIE
(encoding a putative DNA translocase) and
smc
(involved in chromosome structure and partitioning) caused a synthetic lethal phenotype. We constructed a conditional mutation in
smc
and found that many of the
spoIIIE smc
double-mutant cells had a chromosome bisected by a division septum. The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene
spo0J
. These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.
Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of
Streptococcus mutans
Authors: Higuchi Masako , Yamamoto Yuji , Poole Leslie B. , Shimada Mamoru , Sato Yutaka , Takahashi Nobuhiro , Kamio Yoshiyuki
Published: December 2019

We have previously identified two distinct NADH oxidases corresponding to H
2
O
2
-forming oxidase (Nox-1) and H
2
O-forming oxidase (Nox-2) induced in
Streptococcus mutans
. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of
Salmonella typhimurium
alkyl hydroperoxide reductase; an open reading frame upstream of
nox-1
also showed homology to AhpC, the direct peroxide-reducing component of
S. typhimurium
alkyl hydroperoxide reductase. To determine their physiological functions in
S. mutans
, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD
+
but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of
Escherichia coli
TA4315 (
ahpCF
-defective mutant) transformed with pAN119 containing both
nox-1
and
ahpC
genes was not only restored but enhanced relative to that of
E. coli
K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of
S. mutans
to cumene hydroperoxide and H
2
O
2
, implying that the existence of some other antioxidant system(s) independent of Nox-1 in
S. mutans
compensates for the deficiency.
Regulation of
pelD
and
pelE
, Encoding Major Alkaline Pectate Lyases in
Erwinia chrysanthemi
: Involvement of the Main Transcriptional Factors
Authors: Rouanet Carine , Nomura Kinya , Tsuyumu Shinji , Nasser William
Published: December 2019

The main virulence factors of the phytopathogenic bacterium
Erwinia chrysanthemi
are pectinases which attack pectin, the major constituent of the plant cell wall. Of these enzymes, the alkaline isoenzyme named PelD in strain 3937 and PelE in strain EC16 has been described as being particularly important, based on virulence studies of plants. Expression of the
pelD
and
pelE
genes is tightly modulated by various regulators, including the KdgR repressor and the cyclic AMP-cyclic AMP receptor protein (CRP) activator complex. The use of a
lacZ
reporter gene allowed us to quantify the repression of
E. chrysanthemi
3937
pelD
expression exerted by PecS, another repressor of pectinase synthesis. In vitro DNA-protein interaction experiments, centered on the
pelD
and
pelE
wild-type or
pelE
mutated promoter regions, allowed us to define precisely the sequences involved in the binding of these three regulators and of RNA polymerase (RNAP). These studies revealed an unusual binding of the KdgR repressor and suggested the presence of a UP (upstream) element in the
pelD
and
pelE
genes. Investigation of the simultaneous binding of CRP, KdgR, PecS, and the RNAP to the regulatory region of the
pelD
and
pelE
genes showed that (i) CRP and RNAP bind cooperatively, (ii) PecS partially inhibits binding of the CRP activator and of the CRP-RNAP complex, and (iii) KdgR stabilizes the binding of PecS and prevents transcriptional initiation by RNAP. Taken together, our data suggest that PecS attenuates
pelD
and
pelE
expression rather than acting as a true repressor like KdgR. Overall, control of the
pelD
and
pelE
genes of
E. chrysanthemi
appears to be both complex and novel.
The Propanediol Utilization (
pdu
) Operon of
Salmonella enterica
Serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in Coenzyme B
12
-Dependent 1,2-Propanediol Degradation
Authors: Bobik Thomas A. , Havemann Gregory D. , Busch Robert J. , Williams Donna S. , Aldrich Henry C.
Published: December 2019

The propanediol utilization (
pdu
) operon of
Salmonella enterica
serovar Typhimurium LT2 contains genes needed for the coenzyme B
12
-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the
pdu
operon is presented. Analyses of previously unpublished
pdu
DNA sequence substantiated previous studies indicating that the
pdu
operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the
pdu
operon to 21 and the total number of genes at the
pdu
locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that
S. enterica
forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the
pdu
operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B
12
-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.
Plasmid RK2 ParB Protein: Purification and Nuclease Properties
Authors: Johnson Erik P. , Mincer Tracy , Schwab Helmut , Burgin Alex B. , Helinski Donald R.
Published: December 2019

The
parCBA
operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the function of ParC is unknown. In this study ParB was overexpressed by cotranslation with ParC in
Escherichia coli
by using a plasmid construct that contained the
parC
and
parB
genes under the control of the T7 promoter. Purification was achieved by treatment of extracts with Polymin P, followed by ammonium sulfate precipitation and heparin and ion-exchange chromatography. Sizing-column analysis indicated that ParB exists as a monomer in solution. Analysis of the enzymatic properties of purified ParB indicated that the protein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures. ParB also exhibits 5′→3′ exonuclease activity. This ParB activity on a 5′-end-labeled, double-stranded DNA substrate produces a 3′,5′-phosphorylated dinucleotide which is further cleaved to a 3′,5′-phosphorylated mononucleotide. The role of the ParB endonuclease and exonuclease activities in plasmid RK2 stabilization remains to be determined.
Use of Heme-Protein Complexes by the
Yersinia enterocolitica
HemR Receptor: Histidine Residues Are Essential for Receptor Function
Authors: Bracken Charles S. , Baer Michael T. , Abdur-Rashid Asiya , Helms Whitney , Stojiljkovic Igor
Published: December 2019

The abilities of two bacterial active heme transporters, HmbR of
Neisseria meningitidis
and HemR of
Yersinia enterocolitica
, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin-hemoglobin complexes as sources of iron. Expression of functional HemR allowed
Escherichia coli
cells to respond to heme-containing peptides, microperoxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR
H128K
), HemR
H461L
, HemR
H461A
, and HemR
H128A,H461A
mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR
H128K
, HemR
H461A
, and HemR
H461L
. Conversely, the HemR
H192T
, HemR
H352A
, HemR
H352K
, and HemR
H192T,H352K
mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.
Three Asparagine Synthetase Genes of
Bacillus subtilis
Authors: Yoshida Ken-Ichi , Fujita Yasutaro , Ehrlich S. Dusko
Published: December 2019

Three asparagine synthetase genes,
asnB
,
asnH
, and
asnO
(
yisO
), were predicted from the sequence of the
Bacillus subtilis
genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of
asnB
was detected only during exponential growth, that of
asnH
was drastically elevated at the transition between exponential growth and stationary phase, and that of
asnO
was seen only later in sporulation. In a minimal medium, both
asnB
and
asnH
were expressed constitutively during exponential growth and in stationary phase, while the expression of
asnO
was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of
asnH
was partially repressed. Transcription analyses revealed that
asnB
was possibly cotranscribed with a downstream gene,
ytnA
, while the
asnH
gene was transcribed as the fourth gene of an operon comprising
yxbB
,
yxbA
,
yxnB
,
asnH
, and
yxaM
. The
asnO
gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an
Escherichia coli
strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In
B. subtilis
, deletion of
asnO
or
asnH
, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of
asnB
led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that
B. subtilis
might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking
asnO
failed to sporulate, indicating an involvement of this gene in sporulation.