bluuebunny/crossref_metadata_2025_split huggingface dataset:
Poly-β-Hydroxybutyrate Turnover in
Azorhizobium caulinodans
Is Required for Growth and Affects
nifA
Expression
Authors: Mandon Karine , Michel-Reydellet Nathalie , Encarnación Sergio , Kaminski P. Alexandre , Leija Alfonso , Cevallos Miguel A. , Elmerich Claudine , Mora Jaime 
Published: December 2019

ABSTRACT

Azorhizobium caulinodans
is able to fix nitrogen in the free-living state and in symbiosis with the tropical legume
Sesbania rostrata
. The bacteria accumulate poly-β-hydroxybutyrate (PHB) under both conditions. The structural gene for PHB synthase,
phbC
, was inactivated by insertion of an interposon. The mutant strains obtained were devoid of PHB, impaired in their growth properties, totally devoid of nitrogenase activity ex planta (Nif
−
), and affected in nucleotide pools and induced Fix
−
nodules devoid of bacteria. The Nif
−
phenotype was the consequence of the lack of
nifA
transcription. Nitrogenase activity was partially restored to a
phbC
mutant by constitutive expression of the
nifA
gene. However, this constitutive
nifA
expression had no effect on the nucleotide content or on growth of the
phbC
mutant. It is suggested that PHB is required for maintaining the reducing power of the cell and therefore the bacterial growth. These observations also suggest a new control of
nifA
expression to adapt nitrogen fixation to the availability of carbon and reducing equivalents.
Succinoglycan Is Required for Initiation and Elongation of Infection Threads during Nodulation of Alfalfa by
Rhizobium meliloti
Authors: Cheng Hai-Ping , Walker Graham C. 
Published: December 2019

ABSTRACT

Rhizobium meliloti
Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using
R. meliloti
cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of
exo
mutants that are defective or altered in succinoglycan production. Our observations indicate that an
exoY
mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An
exoZ
mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An
exoH
mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the
exoS96
::Tn
5
mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The
exoR95
::Tn
5
mutant, which overproduces succinoglycan to an even greater extent than the
exoS96
::Tn
5
mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.
aguA
, the Gene Encoding an Extracellular α-Glucuronidase from
Aspergillus tubingensis
, Is Specifically Induced on Xylose and Not on Glucuronic Acid
Authors: de Vries Ronald P. , Poulsen Charlotte H. , Madrid Susan , Visser Jaap 
Published: December 2019

ABSTRACT

An extracellular α-glucuronidase was purified and characterized from a commercial
Aspergillus
preparation and from culture filtrate of
Aspergillus tubingensis
. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-
O
-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (
aguA
) was cloned from
A. tubingensis
. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in
A. tubingensis
, and expression was studied in a highly overproducing transformant. The
aguA
gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of
aguA
and also did not modulate the expression on xylose. Addition of glucose prevented expression of
aguA
on xylan but only reduced the expression on xylose.
The Adhesion-Associated
sca
Operon in
Streptococcus gordonii
Encodes an Inducible High-Affinity ABC Transporter for Mn
2+
Uptake
Authors: Kolenbrander Paul E. , Andersen Roxanna N. , Baker Rachel A. , Jenkinson Howard F. 
Published: December 2019

ABSTRACT

ScaA lipoprotein in
Streptococcus gordonii
is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the
scaCBA
operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of
scaC
(ATP-binding protein) or
scaA
(substrate-binding protein) genes resulted in both impaired growth of cells and >70% inhibition of
54
Mn
2+
uptake in media containing <0.5 μM Mn
2+
. In wild-type and
scaC
mutant cells, production of ScaA was induced at low concentrations of extracellular Mn
2+
(<0.5 μM) and by the addition of ≥20 μM Zn
2+
. Sca permease-mediated uptake of
54
Mn
2+
was inhibited by Zn
2+
but not by Ca
2+
, Mg
2+
, Fe
2+
, or Cu
2+
. Reduced uptake of
54
Mn
2+
by
sca
mutants and by wild-type cells in the presence of Zn
2+
was abrogated by the uncoupler carbonylcyanide
m
-chlorophenylhydrazone, suggesting that Mn
2+
uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced >20-fold in
sca
mutants. The addition of 0.1 mM Mn
2+
to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of
sca
mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn
2+
and is essential for growth of streptococci under Mn
2+
-limiting conditions.
Differential Regulation of Multiple Flagellins in
Vibrio cholerae
Authors: Klose Karl E. , Mekalanos John J. 
Published: December 2019

ABSTRACT

Vibrio cholerae
, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum. Motility has been implicated as a virulence determinant in some animal models of cholera, but the relationship between motility and virulence has not yet been clearly defined. We have begun to define the regulatory circuitry controlling motility. We have identified five
V. cholerae
flagellin genes, arranged in two chromosomal loci,
flaAC
and
flaEDB
; all five genes have their own promoters. The predicted gene products have a high degree of homology to each other. A strain containing a single mutation in
flaA
is nonmotile and lacks a flagellum, while strains containing multiple mutations in the other four flagellin genes, including a
flaCEDB
strain, remain motile. Measurement of
fla
promoter-
lacZ
fusions reveals that all five flagellin promoters are transcribed at high levels in both wild-type and
flaA
strains. Measurement of the flagellin promoter-
lacZ
fusions in
Salmonella typhimurium
indicates that the promoter for
flaA
is transcribed by the ς
54
holoenzyme form of RNA polymerase while the
flaE
,
flaD
, and
flaB
promoters are transcribed by the ς
28
holoenzyme. These results reveal that the
V. cholerae
flagellum is a complex structure with multiple flagellin subunits including FlaA, which is essential for flagellar synthesis and is differentially regulated from the other flagellins.
Identification and Characterization of
Escherichia coli
DNA Helicase II Mutants That Exhibit Increased Unwinding Efficiency
Authors: Zhang Gang , Deng Enxin , Baugh Larry , Kushner Sidney R. 
Published: December 2019

ABSTRACT

Using a combination of both ethyl methanesulfonate and site-directed mutagenesis, we have identified a region in DNA helicase II (UvrD) from
Escherichia coli
that is required for biological function but lies outside of any of the seven conserved motifs (T. C. Hodgman, Nature 333:22–23, 1988) associated with the superfamily of proteins of which it is a member. Located between amino acids 403 and 409, alterations in the amino acid sequence DDAAFER lead to both temperature-sensitive and dominant
uvrD
mutations. The
uvrD300
(A406T) and
uvrD301
(A406V) alleles produce UV sensitivity at 44°C but do not affect sensitivity to methyl methanesulfonate (MMS). In contrast, the
uvrD303
mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is dominant to

uvrD
+

when present at six to eight copies per cell. Several of the alleles demonstrated a strong antimutator phenotype. In addition, conjugal recombination is reduced 10-fold in
uvrD303
strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (
uvrD302
) was neutral. To determine the biochemical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a
uvrDΔ294
deletion background and characterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild-type control, while its

K
m

for ATP binding remains unchanged. More importantly, the UvrD303 protein unwinds partial duplex DNA up to 10 times more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propose that the region located between amino acids 403 and 409 serves to regulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA repair systems in which the protein participates.
Specific Amino Acid Substitutions in the Proline-Rich Motif of the
Rhizobium meliloti
ExoP Protein Result in Enhanced Production of Low-Molecular-Weight Succinoglycan at the Expense of High-Molecular-Weight Succinoglycan
Authors: Becker Anke , Pühler Alfred 
Published: December 2019

ABSTRACT

The production of the acidic exopolysaccharide succinoglycan (EPS I) by
Rhizobium meliloti exoP
* mutants expressing an ExoP protein lacking its C-terminal cytoplasmic domain and by mutants characterized by specific amino acid substitutions in the proline-rich motif (RX
4
PX
2
PX
4
SPKX
9
IXGXMXGXG) located from positions 443 to 476 of the ExoP protein was analyzed. The absence of the C-terminal cytoplasmic ExoP domain (positions 484 to 786) and the substitution of both arginine
443
by isoleucine
443
and proline
457
by serine
457
within the proline-rich motif resulted in enhanced production of low-molecular-weight (LMW) EPS I at the expense of high-molecular-weight (HMW) EPS I. The ratios of HMW to LMW EPS I of the wild type and mutant strains increased with osmolarity.
Identification of a Linked Set of Genes in
Serpulina hyodysenteriae
(B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins
Authors: Gabe Jeffrey D. , Dragon Elizabeth , Chang Ray-Jen , McCaman Michael T. 
Published: December 2019

ABSTRACT

A tandem pair of nearly identical genes from
Serpulina hyodysenteriae
(B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of
S. hyodysenteriae
. We have designated these newly identified genes
vspA
and
vspB
(for variable surface protein).
Identification and Characterization of IS
1411
, a New Insertion Sequence Which Causes Transcriptional Activation of the Phenol Degradation Genes in
Pseudomonas putida
Authors: Kallastu Aili , Hõrak Rita , Kivisaar Maia 
Published: December 2019

ABSTRACT

A new insertion sequence (IS element), IS
1411
, was identified downstream of the phenol degradation genes
pheBA
that originated from plasmid DNA of
Pseudomonas
sp. strain EST1001. According to sequence analysis, IS
1411
belongs to a new family of IS elements that has recently been named the ISL
3
family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725–774, 1998). IS
1411
generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS
1411
was discovered as a result of insertional activation of promoterless
pheBA
genes in
Pseudomonas putida
due to the presence of outward-directed promoters at the left end of IS
1411
. Both promoters located on the IS element have sequences that are similar to the consensus sequence of
Escherichia coli
ς
70
. IS
1411
can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.
Nitrogen and Oxygen Regulation of
Bacillus subtilis nasDEF
Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE
Authors: Nakano Michiko M. , Hoffmann Tamara , Zhu Yi , Jahn Dieter 
Published: December 2019

ABSTRACT

The nitrate and nitrite reductases of
Bacillus subtilis
have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by
narGHI
and
nasBC
, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the
nasDE
genes is required for both catabolic and anabolic processes. The
nasDE
genes, together with
nasBC
(encoding assimilatory nitrate reductase) and
nasF
(required for nitrite reductase siroheme cofactor formation), constitute the
nas
operon. Data presented show that transcription of
nasDEF
is driven not only by the previously characterized
nas
operon promoter but also from an internal promoter residing between the
nasC
and
nasD
genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation,
nasDEF
expression and nitrite reductase activity were significantly induced. Anaerobic induction of
nasDEF
required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.
The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in
Methylobacterium extorquens
AM1
Authors: Vorholt Julia A. , Chistoserdova Ludmila , Lidstrom Mary E. , Thauer Rudolf K. 
Published: December 2019

ABSTRACT

An NADP-dependent methylene tetrahydromethanopterin (H
4
MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO
2
in
Methylobacterium extorquens
AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the
mtdA
gene product. The purified enzyme catalyzed the dehydrogenation of methylene H
4
MPT with NADP
+
rather than with NAD
+
, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H
4
F) with NADP
+
. With methylene H
4
F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (
V
max
/

K
m

) were approximately 20-fold lower than with methylene H
4
MPT. Whereas the dehydrogenation of methylene H
4
MPT (E
0
= −390 mV) with NADP
+
(E
0
= −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H
4
F (E
0
= −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from
M. extorquens
AM1 with those of methylene H
4
F dehydrogenases from other bacteria and eucarya and with those of methylene H
4
MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).
Legionella pneumophila
Catalase-Peroxidases: Cloning of the
katB
Gene and Studies of KatB Function
Authors: Bandyopadhyay Purnima , Steinman Howard M. 
Published: December 2019

ABSTRACT

Legionella pneumophila
, the causative organism of Legionnaires’ pneumonia, is spread by aerosolization from man-made reservoirs, e.g., water cooling towers and air conditioning ducts, whose nutrient-poor conditions are conducive to entrance into stationary phase. Exposure to starvation conditions is known to induce several virulence traits in
L. pneumophila
. Since catalase-peroxidases have been extremely useful markers of the stationary-phase response in many bacterial species and may be an avenue for identifying virulence genes in
L. pneumophila
, an investigation of these enzymes was initiated.
L. pneumophila
was shown to contain two bifunctional catalase-peroxidases and to lack monofunctional catalase and peroxidase. The gene encoding the KatB catalase-peroxidase was cloned and sequenced, and
lacZ
fusion and null mutant strains were constructed. Null mutants in
katB
are delayed in the infection and lysis of cultured macrophage-like cell lines. KatB is similar to the KatG catalase-peroxidase of
Escherichia coli
in its 20-fold induction during exponential growth and in playing a role in resistance to hydrogen peroxide. Analysis of the changes in
katB
expression and in the total catalase and peroxidase activity during growth indicates that the 8- to 10-fold induction of peroxidase activity that occurs in stationary phase is attributable to KatA, the second
L. pneumophila
catalase-peroxidase.
Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition
Authors: Gravel Annie , Messier Nancy , Roy Paul H. 
Published: December 2019

ABSTRACT

The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the
attI1
site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.
Characterization of the
mIHF
Gene of
Mycobacterium smegmatis
Authors: Pedulla Marisa L. , Hatfull Graham F. 
Published: December 2019

ABSTRACT

Integration of mycobacteriophage L5 requires the mycobacterial integration host factor (mIHF) in vitro. mIHF is a 105-residue heat-stable polypeptide that is not obviously related to HU or any other small DNA-binding proteins. mIHF is most abundant just prior to entry into stationary phase and is essential for the viability of
Mycobacterium smegmatis.
Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase
Authors: van den Heuvel Robert H. H. , Fraaije Marco W. , Laane Colja , van Berkel Willem J. H. 
Published: December 2019

ABSTRACT

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-
n
-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (
R
)-1-(4′-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171–177, 1998). (
S
)-1-(4′-Hydroxyphenyl)ethanol was found to be a far better substrate than (
R
)-1-(4′-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4′-hydroxyphenyl)alkenes. The relative
cis-trans
stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-
sec
-butylphenol resulted in two (4′-hydroxyphenyl)-
sec
-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.
Identification, Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of
Streptococcus pneumoniae
Penicillin-Binding Protein 1a
Authors: Di Guilmi Anne Marie , Mouz Nicolas , Andrieu Jean-Pierre , Hoskins JoAnn , Jaskunas S. Richard , Gagnon Jean , Dideberg Otto , Vernet Thierry 
Published: December 2019

ABSTRACT

Resistance to β-lactam antibiotics in
Streptococcus pneumoniae
is due to alteration of penicillin-binding proteins (PBPs).
S. pneumoniae
PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in
Escherichia coli
as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.