bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in
Rhodobacter capsulatus
Is Correlated with Cellular Nitrogen Status
Authors: Yakunin Alexander F. , Laurinavichene Tatyana V. , Tsygankov Anatoly A. , Hallenbeck Patrick C. 
Published: December 2019

ABSTRACT

The photosynthetic bacterium
Rhodobacter capsulatus
has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH
4
+
. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH
4
+
, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH
4
+
, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N
2
or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in
R. capsulatus
the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.
Purification of a Glutathione
S
-Transferase and a Glutathione Conjugate-Specific Dehydrogenase Involved in Isoprene Metabolism in
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Kingma Jaap , Kruizinga Wim , Janssen Dick B. 
Published: December 2019

ABSTRACT

A glutathione
S
-transferase (GST) with activity toward 1,2-epoxy-2-methyl-3-butene (isoprene monoxide) and
cis
-1,2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium
Rhodococcus
sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and
cis
-1,2-dichloroepoxyethane, with
V
max
values of 66 and 2.4 μmol min
−1
mg of protein
−1
and

K
m

values of 0.3 and 0.1 mM for isoprene monoxide and
cis
-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM.
1
H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD
+
-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD
+
and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.
Oxygen Depletion-Induced Dormancy in
Mycobacterium bovis
BCG
Authors: Lim Amanda , Eleuterio Marianne , Hutter Bernd , Murugasu-Oei Bernadette , Dick Thomas 
Published: December 2019

ABSTRACT

Gradual depletion of oxygen causes the shift-down of aerobic growing
Mycobacterium bovis
BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and α-crystallin-like protein and is sensitive to metronidazole.
Characterization of
mdcR
, a Regulatory Gene of the Malonate Catabolic System in
Klebsiella pneumoniae
Authors: Peng Hwei-Ling , Shiou Sheng-Ru , Chang Hwan-You 
Published: December 2019

ABSTRACT

The
Klebsiella pneumoniae mdcR
gene, which encodes a LysR-type regulator, was overexpressed in
Escherichia coli
. Purified MdcR was found to bind specifically to the control region of either the malonate decarboxylase (
mdc
) genes or
mdcR
. We have also demonstrated that MdcR is an activator of the expression of the
mdc
genes, whereas it represses the transcription of the putative control region of
mdcR
, P

mdcR

, indicating a negative autoregulatory control.
Genetic Diversity in the Protective Antigen Gene of
Bacillus anthracis
Authors: Price Lance B. , Hugh-Jones Martin , Jackson Paul J. , Keim Paul 
Published: December 2019

ABSTRACT

Bacillus anthracis
is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (
pag
) was sequenced from 26 of the most diverse
B. anthracis
strains to identify potential variation in the toxin and to further our understanding of
B. anthracis
evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of
pag
from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the
pag
corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in
B. anthracis
has occurred with little or no horizontal transfer between the different strains.
Expression of a Germination-Specific Amidase, SleB, of Bacilli in the Forespore Compartment of Sporulating Cells and Its Localization on the Exterior Side of the Cortex in Dormant Spores
Authors: Moriyama Ryuichi , Fukuoka Hideyuki , Miyata Shigeru , Kudoh Sachio , Hattori Atsuhiko , Kozuka Satoshi , Yasuda Yoko , Tochikubo Kunio , Makino Shio 
Published: December 2019

ABSTRACT

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The
sleB
gene encoding this amidase in
Bacillus subtilis
and
Bacillus cereus
was expressed in the forespore compartment of sporulating cells under the control of ς
G
, as shown by Northern blot and primer extension analyses. The forespore-specific expression of
B. subtilis sleB
was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both
Bacillus
species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore’s inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.
Azospirillum irakense
Produces a Novel Type of Pectate Lyase
Authors: Bekri My Ali , Desair Jos , Keijers Veerle , Proost Paul , Searle-van Leeuwen Marjo , Vanderleyden Jos , vande Broek Ann 
Published: December 2019

ABSTRACT

The
pelA
gene from the N
2
-fixing plant-associated bacterium
Azospirillum irakense
, encoding a pectate lyase, was isolated by heterologous expression in
Escherichia coli
. Nucleotide sequence analysis of the region containing
pelA
indicated an open reading frame of 1,296 bp, coding for a preprotein of 432 amino acids with a typical amino-terminal signal peptide of 24 amino acids. N-terminal amino acid sequencing confirmed the processing of the protein in
E. coli
at the signal peptidase cleavage site predicted by nucleotide sequence analysis. Analysis of the amino acid sequence of PelA revealed no homology to other known pectinases, indicating that PelA belongs to a new pectate lyase family. PelA macerates potato tuber tissue, has an alkaline pH optimum, and requires Ca
2+
for its activity. Of several divalent cations tested, none could substitute for Ca
2+
. Methyl-esterified pectin (with a degree of esterification up to 93%) and polygalacturonate can be used as substrates. Characterization of the degradation products formed upon incubation with polygalacturonate indicated that PelA is an endo-pectate lyase generating unsaturated digalacturonide as the major end product. Regulation of
pelA
expression was studied by means of a translational
pelA-gusA
fusion. Transcription of this fusion is low under all growth conditions tested and is dependent on the growth phase. In addition,
pelA
expression was found to be induced by pectin. An
A. irakense pelA
::Tn
5
mutant still displayed pectate lyase activity, suggesting the presence of multiple pectate lyase genes in
A. irakense
.
The Plasmid-Encoded Signal Peptidase SipP Can Functionally Replace the Major Signal Peptidases SipS and SipT of
Bacillus subtilis
Authors: Tjalsma Harold , van den Dolder Juliëtte , Meijer Wilfried J. J. , Venema Gerard , Bron Sierd , van Dijl Jan Maarten 
Published: December 2019

ABSTRACT

The gram-positive eubacterium
Bacillus subtilis
is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the
B. subtilis
plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with
sipP
. These two genes form a conserved structural module of rolling-circle plasmids from
B. subtilis
. As previously shown for the chromosomal
sipS
and
sipT
genes, the transcription of plasmid-borne copies of
sipP
is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of
sipP
starts at the end of exponential growth, about 2 h earlier than that of
sipS
and
sipT
. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.
Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families
Authors: Hill Katja E. , Marchesi Julian R. , Weightman Andrew J. 
Published: December 2019

ABSTRACTDehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial α-halocarboxylic acid (αHA) dehalogenase genes, called group I and group IIdehgenes. The two families are evolutionarily unrelated and together represent almost all of the αHAdehgenes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and IIdehgenes. Amino acid sequences derived from 10 of 11 group Idehpartial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from aRhizobiumsp., had only two of these five residues. Group IIdehgene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinatingl- but notd-2-chloropropionic acid, and derived amino acid sequences for all of the genes exceptdehII°P11showed conservation of previously identified essential residues. Molecular analysis of the twodehfamilies highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group Idehgenes included two putative cryptic or silent genes,dehI°PP3anddehI°17a, produced by different organisms. Group IIdehgenes included two cryptic genes and an active gene,dehIIPP3, that can be switched off and on. All αHA-degrading bacteria so far described wereProteobacteria, a result that may be explained by limitations either in the host range fordehgenes or in isolation methods.
Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the
Bacillus cereus
Group
Authors: Prüß Birgit M. , Francis Kevin P. , von Stetten Felix , Scherer Siegfried 
Published: December 2019

ABSTRACT

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the
Bacillus cereus
group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that
B. cereus
group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.
Molecular and Functional Characterization of the
Rhodopseudomonas palustris
No. 7 Phosphoenolpyruvate Carboxykinase Gene
Authors: Inui Masayuki , Nakata Kaori , Roh Jung Hyeob , Zahn Kenneth , Yukawa Hideaki 
Published: December 2019

ABSTRACT

The
pckA
gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium
Rhodopseudomonas palustris
No. 7. Sequencing of a 2.5-kb chromosomal
Sma
I-
Pst
I fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known
pckA
genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the
pckA
initiation codon and an upstream sequence similar to ς
70
promoters. Studies of a
pckA-lacZ
gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the
pckA
gene was induced in log phase, irrespective of the carbon source. A
R. palustris
No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C
4
-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in
R. palustris
No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.
Intraspecific Diversity of the 23S rRNA Gene and the Spacer Region Downstream in
Escherichia coli
Authors: Antón Ana I. , Martínez-Murcia Antonio J. , Rodríguez-Valera Francisco 
Published: December 2019

ABSTRACT

The molecular microevolution of the 23S rRNA gene (
rrl
) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the
Escherichia coli
ECOR collection. The
rrl
gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (
rrs
) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the
rrs
gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the
rrl
gene seems to be quite similar to that of the
rrs
gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3′ end.
In Vivo Transcription of the
Escherichia coli oxyR
Regulon as a Function of Growth Phase and in Response to Oxidative Stress
Authors: Michán Carmen , Manchado Manuel , Dorado Gabriel , Pueyo Carmen 
Published: December 2019

ABSTRACT

Simultaneous expression of seven genes in
Escherichia coli
was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i)
oxyR
(transcriptional regulator); (ii)
katG
,
dps
,
gorA
, and
ahpCF
(controlled by OxyR); (iii)
sodA
(controlled by SoxRS); and (iv)
trxA
(not related to OxyR or SoxRS). Except for
trxA
, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas
oxyR
,
katG
,
dps
, and
gorA
were activated during exponential growth,
ahpCF
and
sodA
were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for
gorA
and
dps
, respectively. Treatment with H
2
O
2
stimulated expression of the genes (
katG
,
dps
,
ahpCF
, and
gorA
) previously identified as members of the OxyR regulon, except for
oxyR
itself. Induction by H
2
O
2
was a remarkably rapid and reversible process that took place in an OxyR-dependent and ς
S
-independent manner. NaCl induced expression of the genes controlled by OxyR, including the
oxyR
locus. This transcriptional up-regulation was preserved in a strain with the Δ
oxyR
::
kan
mutation, but it was abolished (
ahpCF
) or significantly reduced (
oxyR
and
dps
) in a strain with the
rpoS
::Tn
10
mutation, potentially reflecting positive transcriptional regulation of the
oxyR
regulon by ς
S
. Expression of
trxA
was not increased either by H
2
O
2
stress or by a shift to high-osmolarity conditions.
PhhB, a
Pseudomonas aeruginosa
Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level
Authors: Song Jian , Xia Tianhui , Jensen Roy A. 
Published: December 2019

ABSTRACT

Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in
Pseudomonas aeruginosa
contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in
Escherichia coli
as a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in
trans
. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of
phhB
on
phhA
expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in
E. coli
, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation of
phhB
in
P. aeruginosa
and by comparison of the levels of expression of PhhA in the presence and absence of PhhB in
Escherichia coli
. Experiments using constructs having transcriptional and translational fusions with a
lacZ
reporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of either
l
-tyrosine or
l
-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.
The Periplasmic Nitrate Reductase in
Pseudomonas
sp. Strain G-179 Catalyzes the First Step of Denitrification
Authors: Bedzyk Laura , Wang Tao , Ye Rick W. 
Published: December 2019

ABSTRACT

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in
Pseudomonas
sp. strain G-179, the
nap
gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the
nap
region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap
−
mutant, but that activity could be restored by complementation with the
nap
region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.