bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfate Transport in
Penicillium chrysogenum
: Cloning and Characterization of the
sutA
and
sutB
Genes
Authors: van de Kamp Mart , Pizzinini Enrica , Vos Arnold , van der Lende Ted R. , Schuurs Theo A. , Newbert Roger W. , Turner Geoffrey , Konings Wil N. , Driessen Arnold J. M.
Published: December 2019

In industrial fermentations,
Penicillium chrysogenum
uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes,
sutA
and
sutB
, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative
sB3
mutant of
Aspergillus nidulans
with the
sutB
gene completely restored sulfate uptake activity. The
sutA
gene did not complement the
A. nidulans sB3
mutation, even when expressed under control of the
sutB
promoter. Expression of both
sutA
and
sutB
in
P. chrysogenum
is induced by growth under sulfur starvation conditions. However,
sutA
is expressed to a much lower level than is
sutB
. Disruption of
sutB
resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by
P. chrysogenum
.
Overexpression of a Dominant-Negative Allele of
SEC4
Inhibits Growth and Protein Secretion in
Candida albicans
Authors: Mao Yuxin , Kalb Vernon F. , Wong Brian
Published: December 2019

Candida albicans SEC4
was cloned by complementing the
Saccharomyces cerevisiae sec4-8
mutation, and its deduced protein product (Sec4p) was 63% identical to
S. cerevisiae
Sec4p. One chromosomal
SEC4
allele in
C. albicans
CAI4 was readily disrupted by homologous gene targeting, but efforts to disrupt the second allele yielded no viable null mutants. Although this suggested that
C. albicans SEC4
was essential, it provided no information about this gene’s functions. Therefore, we constructed a mutant
sec4
allele encoding an amino acid substitution (Ser-28→Asn) analogous to the Ser-17→Asn substitution in a
trans
-dominant inhibitor of mammalian Ras protein.
GAL1
-regulated expression plasmids carrying the mutant
sec4
allele (pS28N) had minimal effects in glucose-incubated
C. albicans
transformants, but six of nine transformants tested grew very slowly in galactose. Incubation of pS28N transformants in galactose also inhibited secretion of aspartyl protease (Sap) and caused 90-nm secretory vesicles to accumulate intracellularly, and plasmid curing restored growth and Sap secretion to wild-type levels. These results imply that
C. albicans SEC4
is required for growth and protein secretion and that it functions at a later step in the protein secretion pathway than formation of post-Golgi secretory vesicles. They also demonstrate the feasibility of using inducible dominant-negative alleles to define the functions of essential genes in
C. albicans.
A Periplasmic Location Is Essential for the Role of the ApbE Lipoprotein in Thiamine Synthesis in
Salmonella typhimurium
Authors: Beck Brian J. , Downs Diana M.
Published: December 2019

ApbE is a lipoprotein in
Salmonella typhimurium
, and mutants unable to make this protein have a reduced ability to make thiamine (vitamin B
1
) and require it as a supplement for optimal growth in minimal glucose medium. Polyclonal antibodies specific to ApbE were used to determine that wild-type ApbE is located exclusively in the inner membrane. The periplasmic, monotopic topology of ApbE was determined by using computer-based hydrophobicity plots, LacZ and PhoA gene fusions, and proteinase protection experiments. This extracellular location of ApbE is required for its function, since a cytoplasmic form (ApbE
cyto
) did not allow an
apbE
mutant to grow in the absence of thiamine. A periplasmic form of ApbE (ApbE
peri
) lacking the lipoprotein modification allowed an
apbE
mutant to grow in the absence of thiamine, indicating that soluble ApbE could function in thiamine synthesis and that lipoation and membrane association were not required. Alteration of the amino acid implicated in membrane sorting for other lipoproteins did not result in a relocalization of ApbE to the outer membrane, suggesting that additional sorting determinants exist for ApbE.
Genes of the
sbo-alb
Locus of
Bacillus subtilis
Are Required for Production of the Antilisterial Bacteriocin Subtilosin
Authors: Zheng Guolu , Yan Liang Z. , Vederas John C. , Zuber Peter
Published: December 2019

Bacillus subtilis
JH642 and a wild strain of
B. subtilis
called 22a both produce an antilisterial peptide that can be purified by anion-exchange and gel filtration chromatography. Amino acid analysis confirmed that the substance was the cyclic bacteriocin subtilosin. A mutant defective in production of the substance was isolated from a plasmid gene disruption library. The plasmid insertion conferring the antilisterial-peptide-negative phenotype was located in a seven-gene operon (
alb
, for antilisterial bacteriocin) residing immediately downstream from the
sbo
gene, which encodes the precursor of subtilosin. An insertion mutation in the
sbo
gene also conferred loss of antilisterial activity. Comparison of the presubtilosin and mature subtilosin sequences suggested that certain residues undergo unusual posttranslational modifications unlike those occurring during the synthesis of class I (lantibiotic) or some class II bacteriocins. The putative products of the genes of the operon identified show similarities to peptidases and transport proteins that may function in processing and export. Two
alb
gene products resemble proteins that function in pyrroloquinoline quinone biosynthesis. The use of
lacZ-alb
and
lacZ-sbo
gene fusions, along with primer extension analysis, revealed that the
sbo-alb
genes are transcribed from a major promoter, residing upstream of
sbo
, that is very likely utilized by the ς
A
form of RNA polymerase. The
sbo
and
alb
genes are negatively regulated by the global transition state regulator AbrB and are also under positive autoregulation that is not mediated by the subtilosin peptide but instead requires one or more of the
alb
gene products.
Transcription Regulation of the Colicin K
cka
Gene Reveals Induction of Colicin Synthesis by Differential Responses to Environmental Signals
Authors: Kuhar Irena , Žgur-Bertok Darja
Published: December 2019

Colicin-producing strains occur frequently in natural populations of
Escherichia coli
, and colicinogenicity seems to provide a competitive advantage in the natural habitat. A
cka-lacZ
fusion was used to study the regulation of expression of the colicin K structural gene. Expression is growth phase dependent, with high activity in the late stationary phase. Nutrient depletion induces the expression of
cka
due to an increase in ppGpp. Temperature is a strong signal for
cka
expression, since only basal-level activity was detected at 22°C. Mitomycin C induction demonstrates that
cka
expression is regulated to a lesser extent by the SOS response independently of ppGpp. Increased osmolarity induces a partial increase, while the global regulator integration host factor inhibits expression in the late stationary phase. Induction of
cka
was demonstrated to be independent of the cyclic AMP-Crp complex, carbon source, RpoS, Lrp, H-NS, pH, and short-chain fatty acids. In contrast to colicin E1,
cka
expression is independent of catabolite repression and is partially affected by anaerobiosis only upon SOS induction. These results indicate that while different colicins are expressed in response to some common signals such as nutrient depletion, the expression of individual colicins could be further influenced by specific environmental cues.
Characterization of the
Bradyrhizobium japonicum ftsH
Gene and Its Product
Authors: Narberhaus Franz , Urech Carmen , Hennecke Hauke
Published: December 2019

The
Bradyrhizobium japonicum ftsH
gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the
ftsH
gene in
B. japonicum
failed, suggesting a pivotal cellular function of this gene. The expression of
B. japonicum ftsH
in an
ftsH
-negative
Escherichia coli
strain significantly enhanced the fitness of this mutant and reduced the steady-state level of ς
32
.
PRR1
, a Homolog of
Aspergillus nidulans palF
, Controls pH-Dependent Gene Expression and Filamentation in
Candida albicans
Authors: Porta Amalia , Ramon Ana M. , Fonzi William A.
Published: December 2019

The pH of the environment has been implicated in controlling the yeast-hypha transition and pathogenesis of
Candida albicans
. Several
C. albicans
genes, including
PHR1
and
PHR2
, are pH dependent in their expression. To investigate the mechanism of pH-dependent expression, we have cloned and characterized
PRR1
(for pH response regulator).
PRR1
is homologous to
palF
, a component of the pH response pathway in
Aspergillus nidulans
. Expression of
PRR1
was itself pH dependent, being maximal at acid pH but reduced severalfold at alkaline pH. In a
prr1
null mutant the alkaline-induced expression of
PHR1
was completely abolished. Conversely, expression of
PHR2
was no longer repressed at alkaline pH. A
prr1
null mutant exhibited no morphological abnormalities at either pH; however, it lost the ability to form hyphae on medium 199 and on 10% serum plates. The ability to filament on serum was not restored by forced expression of
PHR1
, indicating that additional
PRR1
-dependent genes are required for hyphal development. These developmental genes appear to be distinct from those controlled by the developmental regulator
EFG1
, since the
EFG1
-dependent gene
HWP1
was expressed normally in the
prr1
null mutant. We conclude that
PRR1
encodes a component of the pH-dependent response pathway in
C. albicans
and that this pathway regulates the expression of multiple components of hyphal development.
Effect of Environmental pH on Morphological Development of
Candida albicans
Is Mediated via the PacC-Related Transcription Factor Encoded by
PRR2
Authors: Ramon Ana M. , Porta Amalia , Fonzi William A.
Published: December 2019

The ability to respond to ambient pH is critical to the growth and virulence of the fungal pathogen
Candida albicans
. This response entails the differential expression of several genes affecting morphogenesis. To investigate the mechanism of pH-dependent gene expression, the
C. albicans
homolog of
pacC
, designated
PRR2
(for pH response regulator), was identified and cloned.
pacC
encodes a zinc finger-containing transcription factor that mediates pH-dependent gene expression in
Aspergillus nidulans
. Mutants lacking
PRR2
can no longer induce the expression of alkaline-expressed genes or repress acid-expressed genes at alkaline pH. Although the mutation did not affect growth of the cells at acid or alkaline pH, the mutants exhibited medium-conditional defects in filamentation.
PRR2
was itself expressed in a pH-conditional manner, and its induction at alkaline pH was controlled by
PRR1
.
PRR1
is homologous to
palF
, a regulator of
pacC
. Thus,
PRR2
expression is controlled by a pH-dependent feedback loop. The results demonstrate that the pH response pathway of
Aspergillus
is conserved and that this pathway has been adapted to control dimorphism in
C. albicans
.
Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of
Escherichia coli
FtsZ
Authors: Ma Xiaolan , Margolin William
Published: December 2019

In
Escherichia coli
, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of
E. coli
FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal
ftsZ
mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an
ftsZ
chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal
ftsZ
mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.
Cloning and Characterization of the
Pseudomonas fluorescens
ATP-Binding Cassette Exporter, HasDEF, for the Heme Acquisition Protein HasA
Authors: Idei Akiko , Kawai Eri , Akatsuka Hiroyuki , Omori Kenji
Published: December 2019

Two ATP-binding cassette (ABC) exporters are present in
Pseudomonas fluorescens
no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The
hasDEF
genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the
Serratia marcescens
ABC exporter Lip system.
P. fluorescens
HasA showed sequence identity of 40 to 49% with HasA proteins from
Pseudomonas aeruginosa
and
Serratia marcescens
. The
P. fluorescens
Has exporter secreted HasA proteins from
P. fluorescens
and
P. aeruginosa
but not
S. marcescens
HasA in
Escherichia coli
, whereas the Has exporter from
S. marcescens
allowed secretion of all three HasA proteins. The
P. fluorescens
HasDEF system also promoted the secretion of the lipase and alkaline protease of
P. fluorescens
. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both
P. fluorescens
and
S. marcescens
sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the
P. fluorescens
Has exporter.
Separate Roles of
Escherichia coli
Replication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA
Authors: Miller Christine , Cohen Stanley N.
Published: December 2019

We report here that the
Escherichia coli
replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive
dnaB
mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of
dnaA
mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.
Conditional Sigma Factor Expression, Using the Inducible Acetamidase Promoter, Reveals that the
Mycobacterium tuberculosis sigF
Gene Modulates Expression of the 16-Kilodalton Alpha-Crystallin Homologue
Authors: Manabe Yukari C. , Chen Jong Min , Ko Chiew G. , Chen Ping , Bishai William R.
Published: December 2019

A chemically inducible acetamidase promoter-
sigF
fusion gene was integrated into the chromosome of
Mycobacterium bovis
BCG. Two-dimensional protein gel analysis permitted the identification of a number of protein spots whose expression was SigF related. One spot upregulated by inappropriate induction of
sigF
expression corresponded to the 16-kDa antigen alpha-crystallin.
A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in
Hansenula polymorpha
DL-1
Authors: Sohn Jung-Hoon , Choi Eui-Sung , Kang Hyun Ah , Rhee Joon-Shick , Rhee Sang-Ki
Published: December 2019

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of
Hansenula polymorpha
DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of
H. polymorpha
. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of
H. polymorpha
with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease
Bal
31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.
Two Roles for the Leucine-Responsive Regulatory Protein in Expression of the Alanine Catabolic Operon (
dadAB
) in
Klebsiella aerogenes
Authors: Janes Brian K. , Bender Robert A.
Published: December 2019

The
lrp
gene, which codes for the leucine-responsive regulatory protein (Lrp), was cloned from
Klebsiella aerogenes
W70. The DNA sequence was determined, and the clone was used to create a disruption of the
lrp
gene. The lack of functional Lrp led to an increased expression of the alanine catabolic operon (
dad
) in the absence of the inducer
l
-alanine but also to a decreased expression of the operon in the presence of
l
-alanine. Thus, Lrp is both a repressor and activator of
dad
expression. Lrp is also necessary for glutamate synthase formation but not for the formation of two other enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate dehydrogenase and histidase.
The bZip Transcription Factor Cap1p Is Involved in Multidrug Resistance and Oxidative Stress Response in
Candida albicans
Authors: Alarco Anne-Marie , Raymond Martine
Published: December 2019

Candida albicans
is an opportunistic pathogenic yeast which frequently develops resistance to the antifungal agent fluconazole (FCZ) in patients undergoing long-term therapy. FCZ-resistant strains often display a reduced intracellular FCZ accumulation which correlates with the overexpression of the ATP-binding cassette transporters
CDR1
and
CDR2
or the major facilitator (MF)
MDR1
. We have recently cloned a
C. albicans
gene, named
CAP1
, which codes for a bZip transcription factor of the AP-1 family homologous to the Yap1 protein involved in multidrug resistance and response to oxidative stress in
Saccharomyces cerevisiae. CAP1
was found to confer FCZ resistance in
S. cerevisiae
by transcriptionally activating
FLR1
, a gene coding for an MF homologous to the
C. albicans MDR1
gene product (A.-M. Alarco, I. Balan, D. Talibi, N. Mainville, and M. Raymond, J. Biol. Chem. 272:19304–19313, 1997). To study the role of
CAP1
in
C. albicans
, we constructed a CAI4-derived mutant strain carrying a homozygous deletion of the
CAP1
gene (CJD21). We found that deletion of
CAP1
did not affect the susceptibility of CJD21 cells to FCZ, cerulenin, brefeldin A, and diamide but caused hypersensitivity to cadmium, 4-nitroquinoline
N
-oxide, 1,10-phenanthroline, and hydrogen peroxide, an effect which was reverted by reintroduction of the
CAP1
gene in these cells. Introduction of a hyperactive truncated allele of
CAP1
(
CAP1-TR
) in CJD21 resulted in resistance of the cells to all of the above compounds except hydrogen peroxide. The hyperresistant phenotype displayed by the CJD21
CAP1-TR
transformants was found to correlate with the overexpression of a number of potential
CAP1
transcriptional targets such as
MDR1
,
CaYCF1
,
CaGLR1
, and
CaTRR1
. Taken together, our results demonstrate that
CAP1
is involved in multidrug resistance and oxidative stress response in
C. albicans
. Finally, disruption of
CAP1
in strain FR2, selected in vitro for FCZ resistance and constitutively overexpressing
MDR1
, did not suppress but rather increased the levels of
MDR1
expression, demonstrating that
CAP1
acts as a negative transcriptional regulator of the
MDR1
gene in FR2 and is not responsible for
MDR1
overexpression in this strain.
Purification and Properties of NADH-Dependent 5,10-Methylenetetrahydrofolate Reductase (MetF) from
Escherichia coli
Authors: Sheppard Christal A. , Trimmer Elizabeth E. , Matthews Rowena G.
Published: December 2019

A K-12 strain of
Escherichia coli
that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15°C and pH 7.2 in a stopped-flow spectrophotometer; the

K
m

for NADH is 13 μM, the

K
m

for CH
2
-H
4
folate is 0.8 μM, and the turnover number under
V
max
conditions estimated for the reaction is 1,800 mol of NADH oxidized min
−1
(mol of enzyme-bound FAD)
−1
. NADPH also serves as a reductant, but exhibits a much higher

K
m

. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from
E. coli
differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen
Clostridium formicoaceticum
and more closely resemble those of the NADH-dependent enzyme from
Peptostreptococcus productus
and the NADPH-dependent enzymes from eukaryotes.
Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium
Pseudomonas syringae
pv. glycinea
Authors: Rohde Bettina H. , Schmid Roland , Ullrich Matthias S.
Published: December 2019

The phytopathogenic bacterium
Pseudomonas syringae
pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of
Pseudomonas putida
M10. The respective
P. syringae
gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated
ncr
for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding
morB
gene of
P. putida
M10. The
ncr
gene was overexpressed in
Escherichia coli
, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the
ncr
gene in different
Pseudomonas
species and various strains of
P. syringae
was analyzed by PCR and Southern blot hybridization. The results indicated that the
ncr
gene is widespread among
P. syringae
pv. glycinea strains but not in other pathovars of
P. syringae
or in any of the other
Pseudomonas
strains tested.
Biochemical and Genetic Evidence for
meta
-Ring Cleavage of 2,4,5-Trihydroxytoluene in
Burkholderia
sp. Strain DNT
Authors: Haigler Billy E. , Johnson Glenn R. , Suen Wen-Chen , Spain Jim C.
Published: December 2019

2,4,5-Trihydroxytoluene (THT) oxygenase from
Burkholderia
sp. strain DNT catalyzes the conversion of THT to an unstable ring fission product. Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction. The THT oxygenase gene (
dntD
) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831–1837, 1993). Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa. The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively. The results suggested that the native protein consists of two identical subunits. The colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site. The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (
meta
-cleavage dioxygenases). In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol. The chemical analysis of the THT ring cleavage product showed that the product was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.
Mycoplasma pneumoniae
Protein P30 Is Required for Cytadherence and Associated with Proper Cell Development
Authors: Romero-Arroyo Cynthia E. , Jordan Jarrat , Peacock Susan J. , Willby Melisa J. , Farmer Mark A. , Krause Duncan C.
Published: December 2019

The attachment organelle of
Mycoplasma pneumoniae
is a polar, tapered cell extension containing an intracytoplasmic, electron-dense core. This terminal structure is the leading end in gliding motility, and its duplication is thought to precede cell division, raising the possibility that mutations affecting cytadherence also confer a defect in motility or cell development. Mycoplasma surface protein P30 is associated with the attachment organelle, and P30 mutants II-3 and II-7 do not cytadhere. In this study, the recombinant wild-type but not the mutant II-3
p30
allele restored cytadherence when transformed into P30 mutants by recombinant transposon delivery. The mutations associated with loss of P30 in mutant II-3 and reacquisition of P30 in cytadhering revertants thereof were identified by nucleotide sequencing of the
p30
gene. Morphological abnormalities that included ovoid or multilobed cells having a poorly defined tip structure were associated with loss of P30. Digital image analysis confirmed quantitatively the morphological differences noted visually. Transformation of the P30 mutants with the wild-type
p30
allele restored a normal morphology, as determined both visually and by digital image analysis, suggesting that P30 plays a role in mycoplasma cell development. Finally, the P30 mutants localized the adhesin protein P1 to the terminal organelle, indicating that P30 is not involved in P1 trafficking but may be required for its receptor-binding function.
Analysis of Phosphorylated Sphingolipid Long-Chain Bases Reveals Potential Roles in Heat Stress and Growth Control in
Saccharomyces
Authors: Skrzypek Marek S. , Nagiec M. Marek , Lester Robert L. , Dickson Robert C.
Published: December 2019

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that
Saccharomyces cerevisiae
cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in
S. cerevisiae
cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25°C, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37°C causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of
DPL1
and
LCB3
, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44°C heat stress 10-fold better than parental cells. These and other data for
dpl1
or
lcb3
single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.
Conditional Stability of the HemA Protein (Glutamyl-tRNA Reductase) Regulates Heme Biosynthesis in
Salmonella typhimurium
Authors: Wang Liying , Elliott Meenal , Elliott Thomas
Published: December 2019

In many bacteria, including the enteric species
Salmonella typhimurium
and
Escherichia coli
, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the
hemA
gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of
S. typhimurium
, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for
E. coli
. The near absence of regulation of
hemA-lac
operon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is ≃20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a
lon clpP
double mutant of
E. coli
. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the
lon clpP
double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.
Impact of either Elevated or Decreased Levels of Cytochrome
bd
Expression on
Shigella flexneri
Virulence
Authors: Way Sing Sing , Sallustio Sandra , Magliozzo Richard S. , Goldberg Marcia B.
Published: December 2019

Shigella
spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that
S. flexneri
cytochrome
bd
expression is necessary for normal intracellular survival and virulence. Cytochrome
bd
is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain.
S. flexneri
mutants that contain a disruption in the
cydC
locus, which leads to defective cytochrome
bd
expression, or in the riboflavin (
ribE
) or ubiquinol-8 (
ubiH
) biosynthetic pathway, which leads to elevated cytochrome
bd
expression, were evaluated in intracellular survival and virulence assays. The
cydC
mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The
ribE
and
ubiH
mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome
bd
is required for
S. flexneri
intracellular survival and virulence.
Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C
4
-Dicarboxylates in
Escherichia coli
Authors: Golby Paul , Davies Suzanne , Kelly David J. , Guest John R. , Andrews Simon C.
Published: December 2019

The
dcuB
gene of
Escherichia coli
encodes an anaerobic C
4
-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition,
dcuB
expression is strongly induced by C
4
-dicarboxylates, suggesting the presence of a novel C
4
-dicarboxylate-responsive regulator in
E. coli
. This paper describes the isolation of a Tn
10
mutant in which the 160-fold induction of
dcuB
expression by C
4
-dicarboxylates is absent. The corresponding Tn
10
mutation resides in the
yjdH
gene, which is adjacent to the
yjdG
gene and close to the
dcuB
gene at ∼93.5 min in the
E. coli
chromosome. The
yjdHG
genes (redesignated
dcuSR
) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the
dcuSR
operon restored the C
4
-dicarboxylate inducibility of
dcuB
expression in the
dcuS
mutant to levels exceeding those of the
dcuS
+
strain by approximately 1.8-fold. The
dcuS
mutation affected the expression of other genes with roles in C
4
-dicarboxylate transport or metabolism. Expression of the fumarate reductase (
frdABCD
) operon and the aerobic C
4
-dicarboxylate transporter (
dctA
) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C
4
-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the
dcuS
mutant was normal. However, under aerobic conditions with C
4
-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a
dctA
mutant. Studies employing a
dcuA dcuB dcuC
triple mutant unable to transport C
4
-dicarboxylates anaerobically revealed that C
4
-dicarboxylate transport is not required for C
4
-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ∼140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C
4
-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of
Klebsiella pneumoniae
. DcuS is not closely related to the C
4
-dicarboxylate-sensing DctS or DctB protein of
Rhodobacter capsulatus
or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.
Analysis of the Type 1 Pilin Gene Cluster
fim
in
Salmonella
: Its Distinct Evolutionary Histories in the 5′ and 3′ Regions
Authors: Boyd E. Fidelma , Hartl Daniel L.
Published: December 2019

The type 1 pilin encoded by
fim
is present in both
Escherichia coli
and
Salmonella
natural isolates, but several lines of evidence indicate that similarities at the
fim
locus may be an example of independent acquisition rather than common ancestry. For example, the
fim
gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the
fim
gene cluster of
Salmonella
, the genes of which show high nucleotide sequence divergence from their
E. coli
counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the
fim
gene cluster is present in all isolates of
S. enterica
but is absent from
S. bongori
. Molecular phylogenetic analyses of the
fimA
and
fimI
genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the
fimZ
,
fimY
, and
fimW
genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the
fimZ
,
fimY
, and
fimW
intergenic regions in the 3′ region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the
fimZ
,
fimY
, and
fimW
genes are less than 46%, which is considerably lower than those of the other six genes of the
fim
cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.
Transcription of the Stationary-Phase-Associated
hspX
Gene of
Mycobacterium tuberculosis
Is Inversely Related to Synthesis of the 16-Kilodalton Protein
Authors: Hu Yanmin , Coates Anthony R. M.
Published: December 2019

The 16-kDa protein, an α-crystallin homologue, is one of the most abundant proteins in stationary-phase
Mycobacterium tuberculosis
. Here, transcription and translation of the
hspX
gene, which encodes the 16-kDa protein, have been investigated by Northern blotting analysis, primer extension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a microaerophilic stationary-phase model. Two transcripts of about 2.5 and 1.1 kb were demonstrated by Northern blot analysis and hybridized to the
hspX
gene probe. Primer extension analysis revealed that the transcription start site is located 33 nucleotides upstream of the
hspX
gene start codon. The cellular level of the
hspX
mRNA was maximum in log-phase bacilli and was markedly reduced after 20 days in unagitated culture, when the organisms had entered the stationary phase. A third transcript of 0.5 kb was detected 0.6 kb downstream of the
hspX
gene; this transcript has a transcriptional pattern completely different from that of the 1.1- and 2.5-kb products, suggesting that there may be another gene in this region. In contrast to the high level of
hspX
mRNA in log-phase bacilli, 16-kDa protein synthesis was low in log-phase bacteria and rose to its maximum after 20 days. In both log-phase and stationary-phase bacteria the mRNA was unstable, with a half-life of 2 min, which indicated that the transcript stability was growth rate independent and not a general means for controlling the gene expression. However, the cellular content of 16-kDa protein, while low in log-phase bacteria, rose to a maximum at 10 days and remained at this high level for up to 50 days, which indicates that this protein is a stable molecule with a low turnover rate. These data suggest that the regulation of
hspX
expression during entry into and maintenance of stationary phase involves translation initiation efficiency and protein stability as potential mechanisms.
Analysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from
Ralstonia eutropha
: Generation of β-Alanine Auxotrophic Tn
5
Mutants and Cloning of the
panD
Gene Region
Authors: Hoppensack Astrid , Rehm Bernd H. A. , Steinbüchel Alexander
Published: December 2019

The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from
Ralstonia eutropha
by 4-phosphopantetheine was investigated. Four β-alanine auxotrophic Tn
5
-induced mutants of
R. eutropha
HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the
panD
gene from
Escherichia coli
, encoding
l
-aspartate-1-decarboxylase (EC
4.1.1.15
), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)
+
transhydrogenase (EC
1.6.1.1
) alpha 1 subunit, encoded by the
pntAA
gene from
Escherichia coli
. The
panD
gene was cloned by complementation of the
panD
mutant of
R. eutropha
Q20. DNA sequencing of the
panD
gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from
E. coli
and
Bacillus subtilis
, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS
−
led to complementation of the
panD
mutants of
R. eutropha
and
E. coli
SJ16, respectively.
panD
-encoded
l
-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of
panD
, an ORF with strong similarity to
pntAA
from
E. coli
, encoding NAD(P)
+
transhydrogenase subunit alpha 1 was found; downstream of
panD
, two ORFs with strong similarity to
pntAB
and
pntB
, encoding subunits alpha 2 and beta of the NAD(P)
+
transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of
pan
and
pnt
genes was found in
R. eutropha
. Labeling experiments using one of the
R. eutropha panD
mutants and [2-
14
C]β-alanine provided no evidence that
R. eutropha
PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the
E. coli panD
mutant exhibit detectable labeling of functional PHA synthase from
R. eutropha
.
Identification and Characterization of SirA, an Iron-Regulated Protein from
Staphylococcus aureus
Authors: Heinrichs Jon H. , Gatlin LaVette E. , Kunsch Charles , Choi Gil H. , Hanson Mark S.
Published: December 2019

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including
Staphylococcus aureus
, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an
S. aureus
genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the
cbr
locus of the plant pathogen
Erwinia chrysanthemi
. The first gene in the locus, which we have designated
sir
for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in
Escherichia coli
. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the
S. aureus
culture medium.
sir
-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.
A Novel Reduced Flavin Mononucleotide-Dependent Methanesulfonate Sulfonatase Encoded by the Sulfur-RegulatedmsuOperon ofPseudomonas aeruginosa
Authors: Kertesz Michael A. , Schmidt-Larbig Karen , Wüest Thomas
Published: December 2019

WhenPseudomonas aeruginosais grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library ofP. aeruginosaPAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have namedmsuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of themsuoperon was analyzed with a transcriptionalmsuD::xylEfusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intactcysBgene, and themsuoperon is therefore part of thecysregulon, since sulfite utilization was found to be CysB independent in this species. Measurements ofmsuD::xylEexpression incysNandcysIgenetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulatingmsuexpression, and sulfonate utilization therefore appears to be tightly regulated.