bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the
n
-Alkane-Assimilating Yeast
Candida tropicalis
Authors: Kanayama Naoki , Ueda Mitsuyoshi , Atomi Haruyuki , Tanaka Atsuo 
Published: December 2019

ABSTRACT

The
n
-alkane-assimilating diploid yeast
Candida tropicalis
possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by
CT-T1A
and
CT-T1B
, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by
CT-T3A
and
CT-T3B
. The physiological functions of these thiolases have been examined by gene disruption. The homozygous
ct-t1aΔ/t1b
Δ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous
ct-t3aΔ/t3b
Δ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on
n
-alkanes (C
10
to C
13
). All thiolase activities in this yeast disappeared with the
ct-t1aΔ/t1b
Δ and
ct-t3aΔ/t3b
Δ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the
CT-T1A
locus in the
ct-t1b
Δ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C
4
substrate butyrate.
Intragenic Suppressors of Induction-Deficient TetR Mutants: Localization and Potential Mechanism of Action
Authors: Biburger Markus , Berens Christian , Lederer Thomas , Krec Traudl , Hillen Wolfgang 
Published: December 2019

ABSTRACT

Eight Tn
10
Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline. The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor. Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction.
Two Separate Regulatory Systems Participate in Control of Swarming Motility of
Serratia liquefaciens
MG1
Authors: Givskov Michael , Östling Jörgen , Eberl Leo , Lindum Peter W. , Christensen Allan Beck , Christiansen Gunna , Molin Søren , Kjelleberg Staffan 
Published: December 2019

ABSTRACT

Swarming motility of
Serratia liquefaciens
MG1 requires the expression of two genetic loci,
flhDC
and
swrI
. Here we demonstrate that the products of the
flhDC
operon (the flagellar master regulator) and the
swrI
gene (the extracellular signal molecule
N
-butanoyl-
l
-homoserine lactone) are global regulators which control two separate regulons.
Contact Stimulation of Tgl and Type IV Pili in
Myxococcus xanthus
Authors: Wall Daniel , Wu Samuel S. , Kaiser Dale 
Published: December 2019

ABSTRACT

Myxococcus xanthus tgl
mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting
tgl
+
cells. The absence of pili in
tgl
mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.
A Functional Homolog of
Escherichia coli
NhaR in
Vibrio cholerae
Authors: Williams S. G. , Carmel-Harel O. , Manning P. A. 
Published: December 2019

ABSTRACT

Escherichia coli
NhaR controls expression of a sodium/proton (Na
+
/H
+
) antiporter, NhaA. The
Vibrio cholerae
NhaR protein shows over 60% identity to those of
Escherichia coli
and
Salmonella enteritidis. V. cholerae
NhaR complements an
E. coli nhaR
mutant for growth in 100 mM LiCl–33 mM NaCl, pH 7.6, and enhances the Na
+
-dependent induction of an
E. coli
chromosomal
nhaA
::
lacZ
fusion. These findings indicate functional homology to
E. coli
NhaR. Two
V. cholerae nhaR
mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of
V. cholerae nhaR
on a low-copy-number plasmid. An
nhaR
mutation had no detectable effect on the virulence of the
V. cholerae
strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.
Mutational Biosynthesis of Novel Rapamycins by a Strain of
Streptomyces hygroscopicus
NRRL 5491 Disrupted in
rapL
, Encoding a Putative Lysine Cyclodeaminase
Authors: Khaw Lake Ee , Böhm Günter A. , Metcalfe Su , Staunton James , Leadlay Peter F. 
Published: December 2019

ABSTRACT

The gene
rapL
lies within the region of the
Streptomyces hygroscopicus
chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into
rapL
by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this
rapL
mutant on media containing added
l
-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that
rapL
encodes a specific
l
-lysine cyclodeaminase important for the production of the
l
-pipecolate precursor. In the presence of added proline derivatives,
rapL
mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.
tRNA
Arg
(
fimU
) and Expression of SEF14 and SEF21 in
Salmonella enteritidis
Authors: Clouthier S. C. , Collinson S. K. , White A. P. , Banser P. A. , Kay W. W. 
Published: December 2019

ABSTRACT

A Tn
10
insertion affecting SEF14 fimbrial synthesis in
Salmonella enteritidis
was located 13 bp upstream of a gene designated
fimU
. The 77-bp DNA sequence of
fimU
from
S. enteritidis
was identical to that of
fimU
encoding tRNA
Arg
(UCU) from
Salmonella typhimurium
and 96% identical to that of the
Escherichia coli argU
homolog. Furthermore, the open reading frame adjacent to and overlapping the 3′ end of
fimU
was similar to the prophage DLP12 integrase gene. The
fimU
-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU. Thus,
fimU
encoded a tRNA
Arg
specific for the rare codon AGA.
fimU
mapped to the SEF21
fim
operon located 15 C’s from the
sef14
gene cluster. Although
fimU
was located within the SEF21
fim
gene cluster, the
fimU
Tn
10
insertion mutant of
S. enteritidis
was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production. SEF17 and SEF18 fimbria production was not affected. Complementation of this mutant with plasmid-borne
fimU
restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21. This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae.
Roles of DnaK and RpoS in Starvation-Induced Thermotolerance of
Escherichia coli
Authors: Rockabrand David , Livers Kevin , Austin Tess , Kaiser Robyn , Jensen Debra , Burgess Richard , Blum Paul 
Published: December 2019

ABSTRACT

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in
Escherichia coli
. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these
dnaK
mutant phenotypes closely resemble those of
rpoS
(ς
38
) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the
dnaK
mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς
32
) suppressors of the
dnaK
mutant with restored levels of RpoS and
dnaK rpoS
double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved
E. coli.
Identification and Characterization of
alcR
, a Gene Encoding an AraC-Like Regulator of Alcaligin Siderophore Biosynthesis and Transport in
Bordetella pertussis
and
Bordetella bronchiseptica
Authors: Beaumont Fiona C. , Kang Ho Young , Brickman Timothy J. , Armstrong Sandra K. 
Published: December 2019

ABSTRACT

A
Bordetella bronchiseptica
iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb
Kpn
I-
Pst
I
Bordetella pertussis
DNA fragment mapping downstream of the alcaligin biosynthesis genes
alcABC
restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation
alcR
. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the
alcR
open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an
Escherichia coli
expression system and visualized in electrophoretic gels. In-frame
alcR
deletion mutants of
B. pertussis
and
B. bronchiseptica
were constructed, and the defined mutants exhibited the
alcR
mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned
alcR
gene provided in
trans
restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of
Bordetella
alcaligin biosynthesis and transport genes and is required for their full expression.
Phosphorylation-Independent Activity of the Response Regulators AlgB and AlgR in Promoting Alginate Biosynthesis in Mucoid
Pseudomonas aeruginosa
Authors: Ma Sheng , Selvaraj Uma , Ohman Dennis E. , Quarless Ryan , Hassett Daniel J. , Wozniak Daniel J. 
Published: December 2019

ABSTRACT

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for
Pseudomonas aeruginosa
in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently,
kinB
was identified downstream of
algB
and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in
kinB
in a mucoid cystic fibrosis isolate,
P. aeruginosa
FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgBΔ1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgBΔ1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni
2+
affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgBΔ1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant
algB
alleles onto the chromosome of a
P. aeruginosa algB
mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation,
P. aeruginosa
strains expressing AlgB.D59N or H-AlgBΔ1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid
P. aeruginosa
appears not to be mediated by conventional phosphorylation-dependent signal transduction.
Cloning and Characterization of Transcription of the
xylAB
Operon in
Thermoanaerobacter ethanolicus
Authors: Erbeznik Milutin , Dawson Karl A. , Strobel Herbert J. 
Published: December 2019

ABSTRACT

The genes encoding xylose isomerase (
xylA
) and xylulose kinase (
xylB
) from the thermophilic anaerobe
Thermoanaerobacter ethanolicus
were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221–227, 1991) promoter region. The bicistronic
xylAB
mRNA was processed by cleavage within the 5′-terminal portion of the XylB-coding sequence. Transcription of
xylAB
was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative
xyl
operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The
T. ethanolicus xylB
gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an
xylB
nucleotide sequence and an
xylAB
operon from a thermophilic anaerobic bacterium.
Assembly of Both the Head and Tail of Bacteriophage Mu Is Blocked in
Escherichia coli groEL
and
groES
Mutants
Authors: Grimaud Régis , Toussaint Ariane 
Published: December 2019

ABSTRACT

Like several other
Escherichia coli
bacteriophages, transposable phage Mu does not develop normally in
groE
hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504–5509, 1987). We show here that lysates obtained upon induction of
groE
Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that
groE
mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.
Salmonella typhimurium
Encodes an SdiA Homolog, a Putative Quorum Sensor of the LuxR Family, That Regulates Genes on the Virulence Plasmid
Authors: Ahmer Brian M. M. , van Reeuwijk Jeroen , Timmers Cynthia D. , Valentine Peter J. , Heffron Fred 
Published: December 2019

ABSTRACT

Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named
sdiA
(suppressor of cell division inhibition), is present in the
Escherichia coli
genome. In this report, we have cloned the
Salmonella typhimurium
homolog of SdiA and performed a systematic screen for
sdiA
-regulated genes. A 4.4-kb fragment encoding the
S. typhimurium sdiA
gene was sequenced and found to encode the 3′ end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (
Salmonella
invasion regulator), and the 5′ end of UvrC. This gene organization is conserved between
E. coli
and
S. typhimurium
. We determined that the
S. typhimurium sdiA
gene was able to weakly complement the
E. coli sdiA
gene for activation of
ftsQAZ
at promoter 2 and for suppression of filamentation caused by an
ftsZ
(Ts) allele. To better understand the function of
sdiA
in
S. typhimurium
, we screened 10,000 random
lacZY
transcriptional fusions (MudJ transposon mutations) for regulation by
sdiA
. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9,
rck
(resistance to complement killing), and ORF11 of the
S. typhimurium
virulence plasmid. The three ORFs have now been named
srgA
,
srgB
, and
srgC
(for
sdiA
-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.
NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in
Klebsiella oxytoca
(
pneumoniae
) M5al
Authors: Wu Qitu , Stewart Valley 
Published: December 2019

ABSTRACT

Klebsiella oxytoca
can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the
nasFEDCBA
operon. We report here the complete
nasFED
sequence. Sequence comparisons indicate that the
nasFED
genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal
nasFED
genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the
nasFED
genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the Δ
nasF
strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.
Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (
cwlJ
) Which Affects Germination of
Bacillus subtilis
Spores
Authors: Ishikawa Shu , Yamane Kunio , Sekiguchi Junichi 
Published: December 2019

ABSTRACT

The predicted amino acid sequence of
Bacillus subtilis ycbQ
(renamed
cwlJ
) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of
B. cereus
and the deduced one of
B. subtilis
. We constructed a
cwlJ::lacZ
fusion in the
B. subtilis
chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the
cwlJ
gene indicated that it is transcribed by Eς
E
RNA polymerase.
cwlJ
-deficient spores responded to both
l
-alanine and AGFK, the
A
580
values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast,
B. subtilis sleB
spores did not release hexosamine at a significant level. While
cwlJ
and
sleB
spores were able to germinate, CJSB (
cwlJ sleB
) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in
A
580
and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of
sleB
mutant spores. The roles of the
sleB
and
cwlJ
mutations in germination and spore maturation are also discussed.
Helicobacter pylori ribBA
-Mediated Riboflavin Production Is Involved in Iron Acquisition
Authors: Worst Dennis J. , M. Gerrits Monique , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G. 
Published: December 2019

ABSTRACT

In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of
Helicobacter pylori
NCTC 11638 which confers to a siderophore synthesis mutant of
Escherichia coli
(EB53
aroB hemA
) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the
ribA
gene of
Bacillus subtilis
. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated
ribBA
) resulted in the formation of one translational product, which was able to complement both the
ribA
and the
ribB
mutation in
E. coli
. Expression of
ribBA
was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in
H. pylori
cells is iron regulated.
E. coli
EB53 containing the plasmid with
H. pylori ribBA
excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by
E. coli
EB53 transformed with the
H. pylori ribBA
gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of
ribBA
in
H. pylori
eliminates its ferric-iron-reduction activity, we conclude that
ribBA
has an important role in ferric-iron reduction and iron acquisition by
H. pylori.