bluuebunny/crossref_metadata_2025 huggingface dataset:
A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis
Authors: Heath Richard J. , Rock Charles O.
Published: December 2019

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX
4
D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the
sn
-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of
Escherichia coli
. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX
4
D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.
The S-Layer Proteins of Two
Bacillus stearothermophilus
Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition
Authors: Egelseer Eva Maria , Leitner Karl , Jarosch Marina , Hotzy Christoph , Zayni Sonja , Sleytr Uwe B. , Sára Margit
Published: December 2019

Two
Bacillus stearothermophilus
wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from
B. stearothermophilus
PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from
B. stearothermophilus
ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from
B. stearothermophilus
ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from
B. stearothermophilus
PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from
B. stearothermophilus
wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.
The
nif
Gene Operon of the Methanogenic Archaeon
Methanococcus maripaludis
Authors: Kessler Peter S. , Blank Carrine , Leigh John A.
Published: December 2019

Nitrogen fixation occurs in two domains,
Archaea
and
Bacteria
. We have characterized a
nif
(nitrogen fixation) gene cluster in the methanogenic archaeon
Methanococcus maripaludis
. Sequence analysis revealed eight genes, six with sequence similarity to known
nif
genes and two with sequence similarity to
glnB
. The gene order,
nifH
,
ORF105
(similar to
glnB
),
ORF121
(similar to
glnB
),
nifD
,
nifK
,
nifE
,
nifN
, and
nifX
, was the same as that found in part in other diazotrophic methanogens and except for the presence of the
glnB
-like genes, also resembled the order found in many members of the
Bacteria
. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the
nif
gene cluster. Northern analysis revealed the presence of either a single 7.6-kb
nif
mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ to
nifH
. Distinctive features of the
nif
gene cluster include the presence of the six primary
nif
genes in a single operon, the placement of the two
glnB
-like genes within the cluster, the apparent physical separation of the cluster from any other
nif
genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens.
Sensory Rhodopsin II Transducer HtrII Is Also Responsible for Serine Chemotaxis in the Archaeon
Halobacterium salinarum
Authors: Hou Shaobin , Brooun Alexei , Suk Yu Hyung , Freitas Tracey , Alam Maqsudul
Published: December 2019

Previously, we demonstrated that the methyl-accepting protein HtrII is the transducer for photoreceptor sensory rhodopsin II. Here, we provide experimental evidence that HtrII is also a chemotransducer. Using an agarose-in-plug bridge method, we show that an HtrII overexpression strain has a quicker response to serine than does an HtrII deletion strain. Furthermore, an in vivo flow assay demonstrates that the deletion strain is unable to modulate methylesterase activity after serine addition or photostimulation, while the overexpression strain shows distinct methanol peaks following both types of stimuli.
The
Saccharomyces cerevisiae SCS2
Gene Product, a Homolog of a Synaptobrevin-Associated Protein, Is an Integral Membrane Protein of the Endoplasmic Reticulum and Is Required for Inositol Metabolism
Authors: Kagiwada Satoshi , Hosaka Kohei , Murata Masayuki , Nikawa Jun-ichi , Takatsuki Akira
Published: December 2019

The
Saccharomyces cerevisiae SCS2
gene has been cloned as a suppressor of inositol auxotrophy of
CSE1
and
hac1/ire15
mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39–45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from
Aplysia californica
(P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580–1583, 1995). In this study we have characterized an
SCS2
gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the
SCS2
gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34°C. Genetic studies reveal that the overexpression of the
INO1
gene rescues the inositol auxotrophy of the
SCS2
disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the
SCS2
disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.
Identification of a Novel
Salmonella
Invasion Locus Homologous to
Shigella ipgDE
Authors: Hong K. Heran , Miller Virginia L.
Published: December 2019

Genes essential for
Salmonella typhimurium
invasion have been localized to
Salmonella
pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn
10
dTc insertion mutants of
S. typhimurium
in the tissue culture invasion assay. One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to the
ipgDEF
operon of the
Shigella flexneri
and
Shigella sonnei
virulence plasmid. A second mutant, SVM168, has an insertion in an IS
3
-type element with homology to the
Salmonella enteritidis
IS
1351
element and
Yersinia enterocolitica
IS
1400
element from a high-pathogenicity island. Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system. In addition, transcription of this operon,
sigDE
(
Salmonella
invasion gene), is dependent on the presence of
sirA
, an activator of
hilA
expression. HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the
sigDE
promoter.
Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in
Escherichia coli
Authors: Ault-Riché Dana , Fraley Cresson D. , Tzeng Chi-Meng , Kornberg Arthur
Published: December 2019

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of
Escherichia coli
mutants revealed that polyP accumulation depends on several regulatory genes,
glnD
(NtrC),
rpoS
,
relA
, and
phoB.
An Exported Inducer Peptide Regulates Bacteriocin Production in
Enterococcus faecium
CTC492
Authors: Nilsen Trine , Nes Ingolf F. , Holo Helge
Published: December 2019

Production of the bacteriocins enterocin A and enterocin B in
Enterococcus faecium
CTC492 was dependent on the presence of an extracellular peptide produced by the strain itself. This induction factor (EntF) was purified, and amino acid sequencing combined with DNA sequencing of the corresponding gene identified it as a peptide of 25 amino acids. The gene encodes a prepeptide of 41 amino acids, including a 16-amino-acid leader peptide of the double-glycine type. Environmental factors influenced the level of bacteriocin production in
E. faecium
CTC492. The optimal pH for bacteriocin production was 6.2. At pH 5.5, growth was slow, and very little bacteriocin was formed. The presence of NaCl or ethanol (EtOH) was also inhibitory to bacteriocin production, and at high concentrations of these solutes, no bacteriocin production was observed. The induction factor induced its own synthesis, and by dilution of the culture 10
6
times or more, nonproducing cultures were obtained. Bacteriocin production was induced in these cultures by addition of EntF. The response was linear, and low bacteriocin production could be induced by about 10
−17
M EntF. This response was attenuated by low pH or the presence of high concentrations of NaCl or EtOH, and 300 times more EntF was needed to induce detectable bacteriocin production in the presence of 6.5% NaCl. High levels of bacteriocin production in cultures grown at low pH or in the presence of high concentrations of NaCl or EtOH were obtained by addition of sufficient amounts of EntF.
A Small Protein (Ags1p) and the Pho80p-Pho85p Kinase Complex Contribute to Aminoglycoside Antibiotic Resistance of the Yeast
Saccharomyces cerevisiae
Authors: Wickert Stephan , Finck Markus , Herz Britta , Ernst Joachim F.
Published: December 2019

We identified the
AGS1
and
AGS3
genes by their ability to partially complement an
ags
mutant (RC1707) which is supersensitive to various aminoglycoside antibiotics (J. F. Ernst and R. K. Chan, J. Bacteriol. 163:8–14, 1985).
AGS1
is located in proximity to the centromere of chromosome III and encodes a small protein of 88 amino acids. The size of the
AGS1
transcript, which in wild-type cells is 1 kb, is reduced to 0.75 kb in mutant RC1707. Disruption of
AGS1
rendered strains supersensitive to hygromycin B and increased their resistance to vanadate. In addition,
ags1
Δ strains underglycosylated invertase but had normal carboxypeptidase Y glycosylation, suggesting that Ags1p is required for the elaboration of outer N-glycosyl chains.
AGS3
was found to be identical to
PHO80
(
TUP7
), which encodes a cyclin activating the Pho85p protein kinase. Deletion of either
PHO80
or
PHO85
led to aminoglycoside supersensitivity;
pho80Δ ags1
Δ strains showed an enhanced-sensitivity phenotype compared to single mutants.
pho80
and
pho85
mutants were rendered resistant by deletion of
PHO4
, indicating that activation of the Pho4p transcription factor is required for increased aminoglycoside sensitivity. Thus, both the Pho80p-Pho85p kinase complex (by Pho4p phosphorylation) and a novel component of the N glycosylation pathway contribute to basal levels of aminoglycoside resistance in
Saccharomyces cerevisiae.
An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to
Lactococcus lactis
Authors: Poquet Isabelle , Ehrlich S. Dusko , Gruss Alexandra
Published: December 2019

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the
Staphylococcus aureus
secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Δ
SP
Nuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in
Lactococcus lactis
, thus demonstrating the suitability of Δ
SP
Nuc to report protein export. The shuttle vector pFUN was designed to construct Δ
SP
Nuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Δ
SP
Nuc to reveal and identify exported proteins was tested by generating an
L. lactis
genomic library in pFUN and by screening for Nuc activity directly in
L. lactis
. All Δ
SP
Nuc fusions displaying a strong Nuc
+
phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in
L. lactis
. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with
L. lactis
show that Δ
SP
Nuc is well suited to report both protein export and membrane protein topology.
The Atrazine Catabolism GenesatzABCAre Widespread and Highly Conserved
Authors: de Souza Mervyn L. , Seffernick Jennifer , Martinez Betsy , Sadowsky Michael J. , Wackett Lawrence P.
Published: December 2019

Pseudomonasstrain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genesatzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous toatzA, -B, and -C. The sequence identities of theatzgenes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.
Evidence for a Regulatory Link of Nitrogen Fixation and Photosynthesis in
Rhodobacter capsulatus
via HvrA
Authors: Kern Monika , Kamp Paul-Bertram , Paschen Annette , Masepohl Bernd , Klipp Werner
Published: December 2019

A
Rhodobacter capsulatus
reporter strain, carrying a constitutively expressed
nifA
gene and a
nifH-lacZ
gene fusion, was used for random transposon Tn
5
mutagenesis to search for genes required for the NtrC-independent ammonium repression of NifA activity. A mutation in
hvrA
, which is known to be involved in low-light activation of the photosynthetic apparatus, released both ammonium and oxygen control of
nifH
expression in this reporter strain, demonstrating a regulatory link of nitrogen fixation and photosynthesis via HvrA. In addition, a significant increase in bacteriochlorophyll
a
(BChl
a
) content was found in cells under nitrogen-fixing conditions. HvrA was not involved in this up-regulation of BChl
a
. Instead, the presence of active nitrogenase seemed to be sufficient for this process, since no increase in BChl
a
content was observed in different
nif
mutants.
Characterization of the
Aspergillus nidulans nmrA
Gene Involved in Nitrogen Metabolite Repression
Authors: Andrianopoulos Alex , Kourambas Sophie , Sharp Julie A. , Davis Meryl A. , Hynes Michael J.
Published: December 2019

The gene
nmrA
of
Aspergillus nidulans
has been isolated and found to be a homolog of the
Neurospora crassa
gene
nmr-1
, involved in nitrogen metabolite repression. Deletion of
nmrA
results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting
areA
gene.
Expression and Regulation of the
sodF
Gene Encoding Iron- and Zinc-Containing Superoxide Dismutase in
Streptomyces coelicolor
Müller
Authors: Kim Eun-Ja , Chung Hye-Jung , Suh Bumsu , Hah Yung Chil , Roe Jung-Hye
Published: December 2019

Streptomyces coelicolor
Müller contains two superoxide dismutases (SODs), nickel-containing (NiSOD) and iron- and zinc-containing SOD (FeZnSOD). The
sodF
gene encoding FeZnSOD was isolated by using PCR primers corresponding to the N-terminal peptide sequence of the purified FeZnSOD and a C-terminal region conserved among known FeSODs and MnSODs. The deduced amino acid sequence exhibited highest similarity to Mn- and FeSODs from
Propionibacterium shermanii
and
Mycobacterium
spp. The transcription start site of the
sodF
gene was determined by primer extension. When the
sodF
gene was cloned in pIJ702 and introduced into
Streptomyces lividans
TK24, it produced at least 30 times more FeZnSOD than the control cells. We disrupted the
sodF
gene in
S. lividans
TK24 and found that the disruptant did not produce any FeZnSOD enzyme activity but produced more NiSOD. The expression of the cloned
sodF
gene in TK24 cells was repressed significantly by Ni, consistent with the regulation pattern in nonoverproducing cells. This finding suggests that the cloned
sodF
gene contains the
cis
-acting elements necessary for Ni regulation. When the
sodF
mRNA in
S. coelicolor
Müller cells was analyzed by S1 mapping of both 5′ and 3′ ends, we found that Ni caused a reduction in the level of monocistronic
sodF
transcripts. Ni did not affect the stability of
sodF
mRNA, indicating that it regulates transcription.
S. lividans
TK24 cells overproducing FeZnSOD became more resistant to oxidants such as menadione and lawsone than the control cells, suggesting the protective role of FeZnSOD. However, the
sodF
disruptant survived as well as the wild-type strain in the presence of these oxidants, suggesting the complementing role of NiSOD increased in the disruptant.
The Periplasmic, Group III Catalase ofVibrio fischeriIs Required for Normal Symbiotic Competence and Is Induced Both by Oxidative Stress and by Approach to Stationary Phase
Authors: Visick Karen L. , Ruby Edward G.
Published: December 2019

The catalase gene,katA, of the sepiolid squid symbiontVibrio fischerihas been cloned and sequenced. The predicted amino acid sequence of KatA has a high degree of similarity to the recently defined group III catalases, including those found inHaemophilus influenzae,Bacteroides fragilis, andProteus mirabilis. Upstream of the predicted start codon ofkatAis a sequence that closely matches the consensus sequence for promoters regulated inEscherichia coliby the alternative sigma factor encoded byrpoS. Further, the level of expression of the clonedkatAgene in anE. coli rpoSmutant is much lower than in wild-typeE. coli. Catalase activity is induced three- to fourfold both as growingV. fischericells approach stationary phase and upon the addition of a small amount of hydrogen peroxide during logarithmic growth. The catalase activity was localized in the periplasm of wild-typeV. fischericells, where its role could be to detoxify hydrogen peroxide coming from the external environment. No significant catalase activity could be detected in akatAnull mutant strain, demonstrating that KatA is the predominately expressed catalase inV. fischeriand indicating thatV. fischericarries only a single catalase gene. The catalase mutant was defective in its ability to competitively colonize the light organs of juvenile squids in coinoculation experiments with the parent strain, suggesting that the catalase enzyme plays an important role in the symbiosis betweenV. fischeriand its squid host.
Generation and Properties of a
Streptococcus pneumoniae
Mutant Which Does Not Require Choline or Analogs for Growth
Authors: Yother Janet , Leopold Klaus , White Johanna , Fischer Werner
Published: December 2019

A mutant (JY2190) of
Streptococcus pneumoniae
Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (
N
-acetylmuramyl-
l
-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86–93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.
PP
i
-Dependent Phosphofructokinase from
Thermoproteus tenax
, an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution
Authors: Siebers Bettina , Klenk Hans-Peter , Hensel Reinhard
Published: December 2019

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC
2.7.1.11
), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP
i
-dependent PFK (PP
i
-PFK; EC
2.7.1.90
), which uses PP
i
instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP
i
-PFK from the anaerobic, hyperthermophilic archaeon
Thermoproteus tenax
, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP
i
-PFK from
T. tenax
consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from
Bacteria
and
Eucarya
; PFK group II contains only PP
i
-PFKs from the genus
Propionibacterium
, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP
i
-dependent enzymes from
T. tenax
and
Amycolatopsis methanolica
and the ATP-PFK from
Streptomyces coelicolor
. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP
i
as a cosubstrate.
Molecular Analysis of the Gene Encoding F
420
-Dependent Glucose-6-Phosphate Dehydrogenase from
Mycobacterium smegmatis
Authors: Purwantini Endang , Daniels Lacy
Published: December 2019

The gene
fgd
, which codes for F
420
-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from
Mycobacterium smegmatis
, and its sequence was determined and analyzed. A homolog of FGD which has a very high similarity to the
M. smegmatis
FGD-derived amino acid sequence was identified in
Mycobacterium tuberculosis
. FGD showed significant homology with F
420
-dependent
N
5
,
N
10
-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from
M. tuberculosis
and
Archaeoglobus fulgidus
, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases. Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences. Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences. Moderately high levels of FGD were expressed in
Escherichia coli
BL21(DE3) carrying
fgd
in pBluescript.
Involvement of Superoxide Dismutase in Spore Coat Assembly in
Bacillus subtilis
Authors: Henriques Adriano O. , Melsen Lawrence R. , Moran Charles P.
Published: December 2019

Endospores of
Bacillus subtilis
are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the
sodA
locus.
sodA
is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from
B. subtilis
. Disruption of the
sodA
locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The
sodA
mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of
sodA
mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the
cotG
gene in sporulating cells of the
sodA
mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a
sodA
mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a
cotG
mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer.
Nucleotide Sequence and Spatiotemporal Expression of the
Vibrio cholerae vieSAB
Genes during Infection
Authors: Lee Sang Ho , Angelichio Michael J. , Mekalanos John J. , Camilli Andrew
Published: December 2019

The
iviVII
gene of
Vibrio cholerae
was previously identified by a screen for genes induced during intestinal infection. In the present study, nucleotide sequence analysis revealed that
iviVII
is a 1,659-bp open reading frame, herein designated
vieB
, that is predicted to be last in a tricistronic operon (
vieSAB
). The deduced amino acid sequence of VieS exhibited similarity to the sensor kinase component, and those of VieA and VieB were similar to the response regulator components, respectively, of the two-component signal transduction family. Analysis of transcriptional fusions to a site-specific DNA recombinase reporter,
tnpR
, revealed that
vieS
and
vieA
are transcribed during in vitro growth in a
vieAB
-independent and
vieA
-dependent manner, respectively. In contrast, transcription of
vieB
occurred exclusively during infection and was not dependent upon VieB. We conclude that the
vieSAB
genes are differentially regulated, at least during laboratory growth. Use of a
V. cholerae
strain harboring a
vieB
::
tnpR
transcriptional fusion allowed the kinetics and location of
vieB
expression within the intestine to be determined. We found that
vieB
transcription is induced shortly after infection of the proximal and mid-small intestine.
2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by
Rhodopseudomonas palustris
Authors: Pelletier Dale A. , Harwood Caroline S.
Published: December 2019

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium
Rhodopseudomonas palustris
by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the
badI
gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of
badI
indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min
−1
mg of protein
−1
. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An
R. palustris
mutant, carrying a chromosomal disruption of
badI
, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by
R. palustris.
Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems
Authors: Parales Rebecca E. , Emig Matthew D. , Lynch Nancy A. , Gibson David T.
Published: December 2019

Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (α and β). To assess the contributions of the α and β subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids inEscherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different β subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.
Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites
Authors: Chugani Sudha A. , Parsek Matthew R. , Chakrabarty A. M.
Published: December 2019

ThecatBCAoperon ofPseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule,cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCApromoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCApromoter within thecatBstructural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to thecatBtranscriptional start site and lies within thecatBopen reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZtranscriptional fusions in vivo. Our results suggest a probablecis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within thecatBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.
A Low pH-Inducible, PhoPQ-Dependent Acid Tolerance Response Protects
Salmonella typhimurium
against Inorganic Acid Stress
Authors: Bearson Bradley L. , Wilson Lee , Foster John W.
Published: December 2019

The acid tolerance response enables
Salmonella typhimurium
to survive exposures to potentially lethal acidic environments. The acid stress imposed in a typical assay for acid tolerance (log-phase cells in minimal glucose medium) was shown to comprise both inorganic (i.e., low pH) and organic acid components. A gene previously determined to affect acid tolerance,
atbR
, was identified as
pgi
, the gene encoding phosphoglucoisomerase. Mutations in
pgi
were shown to increase acid tolerance by preventing the synthesis of organic acids. Protocols designed to separate the stresses of inorganic from organic acids revealed that the regulators ς
38
(RpoS), Fur, and Ada have major effects on tolerance to organic acid stress but only minor effects on inorganic acid stress. In contrast, the two-component regulatory system PhoP (identified as acid shock protein ASP29) and PhoQ proved to be important for tolerance to organic acid stress but had little effect against organic acid stress. PhoP mutants also failed to induce four ASPs, confirming a role for this regulator in acid tolerance. Acid shock induction of PhoP appears to occur at the transcriptional level and requires the PhoPQ system. Furthermore, induction by acid occurs even in the presence of high concentrations of magnesium, the ion known to be sensed by PhoQ. These results suggest that PhoQ can sense both Mg
2+
and pH. Since
phoP
mutants are avirulent, the low pH activation of this system has important implications concerning the pathogenesis of
S. typhimurium
. The involvement of four regulators, two of which are implicated in virulence, underscores the complexity of the acid tolerance stress response and further suggests that features of acid tolerance and virulence are interwoven.
A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of
Pseudomonas pseudoalcaligenes
JS45
Authors: He Zhongqi , Spain Jim C.
Published: December 2019

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by
Pseudomonas pseudoalcaligenes
JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The

K
m

for 2-aminomuconate was approximately 67 μM, and the
V
max
was 125 μmol · min
−1
· mg
−1
. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.
A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in
Pseudomonas
sp. Strain U2
Authors: Fuenmayor Sergio L. , Wild Mark , Boyes Alastair L. , Williams Peter A.
Published: December 2019

Pseudomonas
sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb
Bam
HI fragment. The genes for the naphthalene dioxygenase genes
nagAa
(for ferredoxin reductase),
nagAb
(for ferredoxin), and
nagAc
and
nagAd
(for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for
nagB
(for naphthalene
cis
-dihydrodiol dehydrogenase) and
nagF
(for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between
nagAa
and
nagAb
were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant
Escherichia coli
strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated
nagG
, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated
nagH
, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in
E. coli
,
nagG
,
nagH
, and
nagAb
, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of
nagG
,
nagH
, and
nagAb
demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although
E. coli
appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order
nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF
represents the archetype for naphthalene strains which use the gentisate pathway rather than the
meta
cleavage pathway of catechol.
Purification and Biochemical Characterization of the Lambda Holin
Authors: Smith David L. , Struck Douglas K. , Scholtz J. Martin , Young Ry
Published: December 2019

Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the λ holin, and one of the two products of gene
S
is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated
S105H94
, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an
S
mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.
Regulation of a New Cell Wall Hydrolase Gene,
cwlF
, Which Affects Cell Separation in
Bacillus subtilis
Authors: Ishikawa Shu , Hara Yoshiko , Ohnishi Ryo , Sekiguchi Junichi
Published: December 2019

Bacillus subtilis
produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from
B. subtilis cwlB sigD
mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the
papQ
gene product. Disruption of the
papQ
gene in the
B. subtilis
chromosome led to the complete loss of CwlF, indicating that
papQ
is identical to
cwlF
. CwlF exhibits high sequence similarity to the p60 proteins of
Listeria
species, NlpC proteins of
Escherichia coli
and
Haemophilus influenzae
, and Enp2 protein of
Bacillus sphaericus
. The β-galactosidase activity of the
cwlF-lacZ
transcriptional fusion and Northern blot analysis of the
cwlF
gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the
cwlF
gene is transcribed mainly by EςA RNA polymerase and weakly by EςH RNA polymerase. While the cells of the
cwlF
-deficient mutant were about twice as long as those of the wild-type strain, the
cwlF sigD
double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the
sigD
mutant. The CwlF production was not affected by the pleiotropic mutations
flaD1
and
degU32
(Hy), which endow cells with the ability of extensive filamentation.
Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon
Sulfolobus shibatae
Constrain Negative DNA Supercoils
Authors: Mai Viet Q. , Chen Xulin , Hong Ray , Huang Li
Published: December 2019

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon
Sulfolobus shibatae
. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from
Sulfolobus acidocaldarius
and
Sulfolobus solfataricus
in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from
S. shibatae
is relaxed, suggest that regions of free DNA in the
S. shibatae
genome, if present, are highly positively supercoiled.
Identification and Enzymatic Characterization of the Maltose-Inducible α-Glucosidase MalL (Sucrase-Isomaltase-Maltase) of
Bacillus subtilis
Authors: Schönert Stefan , Buder Thomas , Dahl Michael K.
Published: December 2019

A gene coding for a putative α-glucosidase has been identified in the open reading frame
yvdL
(now termed
malL
), which was sequenced as part of the
Bacillus subtilis
genome project. The enzyme was overproduced in
Escherichia coli
and purified. Further analyses indicate that MalL is a specific oligo-1,4-1,6-α-glucosidase (sucrase-maltase-isomaltase). MalL expression in
B. subtilis
requires maltose induction and is subject to carbon catabolite repression by glucose and fructose. Insertional mutagenesis of
malL
resulted in a complete inactivation of the maltose-inducible α-glucosidase activity in crude protein extracts and a Mal
−
phenotype.