bluuebunny/crossref_metadata_2025 huggingface dataset:
IS
481
and IS
1002
of
Bordetella pertussis
Create a 6-Base-Pair Duplication upon Insertion at a Consensus Target Site
Authors: Stibitz Scott
Published: December 2019

The insertion sequence IS
481
and its isoform IS
1002
have been observed to transpose into the
bvgAS
locus of
Bordetella pertussis
, for which the DNA sequence has previously been determined. Upon insertion of IS
481
at three different sites and IS
1002
at one site, a 6-bp sequence originally present was found at the junction of
bvg
and insertion sequence DNA. This indicates that, contrary to prior reports, IS
481
and IS
1002
do create a duplication upon insertion. In this light, examination of these and other examples of IS
481
and IS
1002
reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS
481
or IS
1002
, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.
Limonene-1,2-Epoxide Hydrolase from
Rhodococcus erythropolis
DCL14 Belongs to a Novel Class of Epoxide Hydrolases
Authors: van der Werf Mariët J. , Overkamp Karin M. , de Bont Jan A. M.
Published: December 2019

An epoxide hydrolase from
Rhodococcus erythropolis
DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when
R. erythropolis
is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50°C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4′-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1,10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.
Mutation Analysis of PobR and PcaU, Closely Related Transcriptional Activators in
Acinetobacter
Authors: Kok Ruben G. , D’Argenio David A. , Ornston L. Nicholas
Published: December 2019

Acinetobacter
PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite
p
-hydroxybenzoate and activates transcription of
pobA
, the structural gene for the enzyme that converts
p
-hydroxybenzoate to protocatechuate. This compound, differing from
p
-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from
Acinetobacter
strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which
pobR
function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the
pobA-pobR
intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on
p
-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Transcriptional Regulation of the
Streptococcus mutans gal
Operon by the GalR Repressor
Authors: Ajdić Dragana , Ferretti Joseph J.
Published: December 2019

The galactose operon of
Streptococcus mutans
is transcriptionally regulated by a repressor protein (GalR) encoded by the
galR
gene, which is divergently oriented from the structural genes of the
gal
operon. To study the regulatory function of GalR, we partially purified the protein and examined its DNA binding activity by gel mobility shift and DNase I footprinting experiments. The protein specifically bound to the
galR-galK
intergenic region at an operator sequence, the position of which would suggest that GalR plays a role in the regulation of the
gal
operon as well as autoregulation. To further examine this hypothesis, transcriptional start sites of the
gal
operon and the
galR
gene were determined. Primer extension analysis showed that both promoters overlap the operator, indicating that GalR most likely represses transcription initiation of both promoters. Finally, the results from in vitro binding experiments with potential effector molecules suggest that galactose is a true intracellular inducer of the galactose operon.
Subcellular Localization of
Bacillus subtilis
SMC, a Protein Involved in Chromosome Condensation and Segregation
Authors: Graumann Peter L. , Losick Richard , Strunnikov Alexander V.
Published: December 2019

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium
Bacillus subtilis
. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the
B. subtilis
cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated
smc
gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.
Roles of Multiple Promoters in Transcription of Ribosomal DNA: Effects of Growth Conditions on Precursor rRNA Synthesis in Mycobacteria
Authors: Gonzalez-y-Merchand J. A. , Colston M. J. , Cox R. A.
Published: December 2019

The roles of multiple promoters in the synthesis of rRNA under different conditions of growth were investigated, using two mycobacterial species as model organisms. When
Mycobacterium smegmatis
was grown under optimal conditions, its two rRNA operons contributed equally, with two promoters, one from each operon, being responsible for most transcripts. In stationary-phase growth or balanced growth under carbon starvation conditions, one operon (
rrnA
f
) dominated and its three promoters contributed more equally to the generation of transcripts.
Mycobacterium tuberculosis
has a single operon with two promoters, one of which generated 80% of transcripts, at all stages of growth. We infer that each promoter functions independently according to its intrinsic strength when cells are growing slowly so that one operon with three promoters is roughly equivalent to three operons with one promoter; at high growth rates, occlusion effects reduce the efficiency of multiple promoters to that of a single promoter.
Expression of Alkane Hydroxylase from
Acinetobacter
sp. Strain ADP1 Is Induced by a Broad Range of n- Alkanes and Requires the Transcriptional Activator AlkR
Authors: Ratajczak Andreas , Geißdörfer Walter , Hillen Wolfgang
Published: December 2019

In
Acinetobacter
sp. strain ADP1, alkane degradation depends on at least five essential genes.
rubAB
and
xcpR
are constitutively transcribed. Here we describe inducible transcription of
alkM
, which strictly depends on the presence of the transcriptional activator AlkR.
alkR
itself is expressed at a low level, while a chromosomally located
alkM
::
lacZ
fusion is inducible by middle-chain-length alkanes from heptane to undecane, which do not support growth of ADP1, and by long-chain-length alkanes from dodecane to octadecane, which are used as sources of carbon and energy. The putative AlkM substrate 1-dodecene is also an effective inducer. Products of alkane hydroxylase activity like 1-dodecanol prevent induction of
alkM
expression.
alkM
is expressed only in stationary phase, suggesting its dependence on at least one other regulatory mechanism.
Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls
Authors: Hurtubise Yves , Barriault Diane , Sylvestre Michel
Published: December 2019

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in
Comamonas testosteroni
B-356 and in
Pseudomonas
sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP
BPH
), a ferredoxin (FER
BPH
), and a ferredoxin reductase (RED
BPH
). B-356 BPH dox preferentially catalyzes the oxidation of the double-
meta
-substituted congener 3,3′-dichlorobiphenyl over the double-
para
-substituted congener 4,4′-dichlorobiphenyl or the double-
ortho
-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent
meta-para
carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the
meta-para
dioxygenation of chimeric enzymes obtained by exchanging the ISP
BPH
α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP
BPH
of strain LB400 and the β subunit of ISP
BPH
of strain B-356 (the α
LB400
β
B-356
chimera) and the α
B-356
β
LB400
chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP
BPH
β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α
B-356
β
LB400
chimera should have behaved like B-356 ISP
BPH
; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP
BPH
. On the other hand, the α
LB400
β
B-356
chimera showed features of both B-356 and LB400 ISP
BPH
where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the
meta-para
oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.
atp
Mutants of
Escherichia coli
Fail To Grow on Succinate Due to a Transport Deficiency
Authors: Boogerd Fred C. , Boe Lars , Michelsen Ole , Jensen Peter Ruhdal
Published: December 2019

Escherichia coli atp
mutants, which lack a functional H
+
-ATPase complex, are capable of growth on glucose but not on succinate or other C
4
-dicarboxylates (Suc
−
phenotype). Suc
+
revertants of an
atp
deletion strain were isolated which were capable of growth on succinate even though they lack the entire H
+
-ATPase complex. Complementation in
trans
with the
yhiF
gene suppressed the growth of the Suc
+
mutants on succinate, which implicates the
yhiF
gene product in the regulation of C
4
-dicarboxylate metabolism. Indeed, when the
E. coli
C
4
-dicarboxylate transporter (encoded by the
dctA
gene) was expressed in
trans
, the Suc
−
phenotype of the
atp
deletion strain reverted to Suc
+
, which shows that the reason why the
E. coli atp
mutant is unable to grow aerobically on C
4
-dicarboxylates is insufficient transport capacity for these substrates.
A
Saccharomyces cerevisiae
Mutant Lacking a K
+
/H
+
Exchanger
Authors: Ramírez Jorge , Ramírez Oscar , Saldaña Carlos , Coria Roberto , Peña Antonio
Published: December 2019

The
KHA1
gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K
+
/H
+
antiporter (873 amino acids) in
Saccharomyces cerevisiae
was disrupted by homologous recombination. The core protein is similar to the putative Na
+
/H
+
antiporters from
Enterococcus hirae
(
NAPA
gene) and
Lactococcus lactis
(
LLUPP
gene) and the putative K
+
/H
+
exchanger from
Escherichia coli
(
KEFC
gene). Disruption of the
KHA1
gene resulted in an increased K
+
accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of
86
Rb
+
uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K
+
accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.
Accumulation of the Enterobacterial Common Antigen Lipid II Biosynthetic Intermediate Stimulates
degP
Transcription in
Escherichia coli
Authors: Danese Paul N. , Oliver George R. , Barr Kathleen , Bowman Gregory D. , Rick Paul D. , Silhavy Thomas J.
Published: December 2019

In
Escherichia coli
, transcription of the
degP
locus, which encodes a heat-shock-inducible periplasmic protease, is controlled by two parallel signal transduction systems that each monitor extracytoplasmic protein physiology. For example, the heat-shock-inducible sigma factor, ς
E
, controls
degP
transcription in response to the overproduction and folded state of various extracytoplasmic proteins. Similarly, the CpxA/R two-component signal transduction system increases
degP
transcription in response to the overproduction of a variety of extracytoplasmic proteins. Since
degP
transcription is attuned to the physiology of extracytoplasmic proteins, we were interested in identifying negative transcriptional regulators of
degP
. To this end, we screened for null mutations that increased transcription from a strain containing a
degP-lacZ
reporter fusion. Through this approach, we identified null mutations in the
wecE
,
rmlA
ECA
, and
wecF
loci that increase
degP
transcription. Interestingly, each of these loci is responsible for synthesis of the enterobacterial common antigen (ECA), a glycolipid situated on the outer leaflet of the outer membrane of members of the family
Enterobacteriaceae
. However, these null mutations do not stimulate
degP
transcription by eliminating ECA biosynthesis. Rather, the
wecE
,
rmlA
ECA
, and
wecF
null mutations each impede the same step in ECA biosynthesis, and it is the accumulation of the ECA biosynthetic intermediate, lipid II, that causes the observed perturbations. For example, the lipid II-accumulating mutant strains each (i) confer upon
E. coli
a sensitivity to bile salts, (ii) confer a sensitivity to the synthesis of the outer membrane protein LamB, and (iii) stimulate both the Cpx pathway and ς
E
activity. These phenotypes suggest that the accumulation of lipid II perturbs the structure of the bacterial outer membrane. Furthermore, these results underscore the notion that although the Cpx and ς
E
systems function in parallel to regulate
degP
transcription, they can be simultaneously activated by the same perturbation.
Adherence of the Gram-Positive Bacterium
Ruminococcus albus
to Cellulose and Identification of a Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family of Proteins
Authors: Pegden Randall S. , Larson Marilynn A. , Grant Richard J. , Morrison Mark
Published: December 2019

The adherence of
Ruminococcus albus
8 to crystalline cellulose was studied, and an affinity-based assay was also used to identify candidate cellulose-binding protein(s). Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both phenylacetic and phenylpropionic acids in the growth medium, and the addition of carboxymethylcellulose (CMC) to assays decreased the adherence of the bacterium to cellulose. A cellulose-binding protein with an estimated molecular mass following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ∼21 kDa, designated CbpC, was present in both cellobiose- and cellulose-grown cultures, and the relative abundance of this protein increased in response to growth on cellulose. Addition of 0.1% (wt/vol) CMC to the binding assays had an inhibitory effect on CbpC binding to cellulose, consistent with the notion that CbpC plays a role in bacterial attachment to cellulose. The nucleotide sequence of the
cbpC
gene was determined by a combination of reverse genetics and genomic walking procedures. The
cbpC
gene encodes a protein of 169 amino acids with a calculated molecular mass of 17,655 Da. The amino-terminal third of the CbpC protein possesses the motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in gram-negative, pathogenic bacteria. The remainder of the predicted CbpC sequence was found to have significant identity with 72- and 75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen protein of
Rickettsia
spp., as well as one of the major capsid glycoproteins of the
Chlorella
virus PBCV-1. Northern blot analysis showed that phenylpropionic acid and ruminal fluid increase
cbpC
mRNA abundance in cellobiose-grown cells. These results suggest that CbpC is a novel cellulose-binding protein that may be involved in adherence of
R. albus
to substrate and extends understanding of the distribution of the Pil family of proteins in gram-positive bacteria.
Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in
Methanothermus fervidus
Authors: Matussek Karl , Moritz Patrick , Brunner Nina , Eckerskorn Christoph , Hensel Reinhard
Published: December 2019

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon
Methanothermus fervidus
, sequenced the coding gene, and expressed it in
Escherichia coli
. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in
M. fervidus
, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the
V
max
of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from
E. coli
revealed a twofold-higher
V
max
of the enzyme from
M. fervidus
in the synthesizing direction.
Role of the
parCBA
Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance
Authors: Easter Carla L. , Schwab Helmut , Helinski Donald R.
Published: December 2019

The
par
region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,
parCBA
and
parDE
, and a
cis
-acting site.
parDE
encodes a postsegregational killing system, and
parCBA
encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the
parCBA
region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of
Escherichia coli
and two strains of
Pseudomonas aeruginosa
. The intact
parCBA
operon was effective in stabilizing a conjugation-defective RK2 derivative in
E. coli
MC1061K and RR1 but was relatively ineffective in
E. coli
MV10Δlac. In the two strains in which the
parCBA
operon was effective, deletions in
parB
,
parC
, or both
parB
and
parC
caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the
parA
gene resulted in a much greater loss of
parCBA
activity. For
P. aeruginosa
PAO1161Rif
r
, the
parCBA
operon provided little if any plasmid stability, but for
P. aeruginosa
PAC452Rif
r
, the RK2 plasmid was stabilized to a substantial extent by
parCBA
. With this latter strain,
parA
and
res
alone were sufficient for stabilization. The
cer
resolvase system of plasmid ColE1 and the
loxP
/Cre system of plasmid P1 were tested in comparison with the
parCBA
operon. We found that, not unlike what was previously observed with MC1061K,
cer
failed to stabilize the RK2 plasmid with
par
deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The
loxP
/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three
E. coli
strains. These observations indicate that the
parA
gene, along with its
res
site, exhibits a significant level of plasmid stabilization in the absence of the
parC
and
parB
genes but that in at least one
E. coli
strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with
parCBA
or the
cer
and
loxP
/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.