bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anaerobic Regulation of
Bacillus subtilis
Krebs Cycle Genes
Authors: Nakano Michiko M. , Zuber Peter , Sonenshein Abraham L. 
Published: December 2019

ABSTRACT

Krebs cycle enzyme activity in
Bacillus subtilis
was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions. The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures. These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription. This repression was not dependent on either the
fnr
or
resDE
gene products, which have been shown to regulate expression of other
B. subtilis
genes in response to anaerobic conditions. Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression. A dyad symmetry element located between positions −73 and −59 relative to the transcription start site of the aconitase gene (
citB
) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth. The deletion of the upstream arm of the dyad symmetry region abolished the
citB
repression observed during anaerobic growth. Furthermore, neither
citZ
or
citB
was repressed in an anaerobically grown
citB
mutant, an effect that was very likely the result of citrate accumulation. These results suggest that catabolite repression and anaerobic repression of
citZ
and
citB
are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.
Aeromonas hydrophila
Adenylyl Cyclase 2: a New Class of Adenylyl Cyclases with Thermophilic Properties and Sequence Similarities to Proteins from Hyperthermophilic Archaebacteria
Authors: Sismeiro Odile , Trotot Pascale , Biville Francis , Vivares Christian , Danchin Antoine 
Published: December 2019

ABSTRACT

Complementation of an
Escherichia coli cya
mutant with a genomic library from
Aeromonas hydrophila
allowed isolation of clones containing two different
cya
genes. Whereas one of these genes (
cyaA
) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (
cyaB
) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65°C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in
A. hydrophila
, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to
cyaA
, the
cyaB
gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the
cyaB
gene in a
cyaA
mutant, as well as in a
cyaA cyaB
mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria:
Methanobacterium thermoautotrophicum
,
Archaeglobus fulgidus
, and
Methanococcus jannaschii
. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.
Copper-Binding Compounds from
Methylosinus trichosporium
OB3b
Authors: DiSpirito Alan A. , Zahn James A. , Graham David W. , Kim Hyung J. , Larive Cynthia K. , Derrick Tiffany S. , Cox Charles D. , Taylor Alan 
Published: December 2019

ABSTRACT

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO
C
) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of
Methylosinus trichosporium
OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L
1
and CBC-L
2
, respectively. The amino acid sequence of CBC-L
1
is S?MYPGS?M, and that of CBC-L
2
is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L
1
showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g
⊥
= 2.087, g
∥︀
= 2.42 G, ‖A
∥︀
‖ = 128 G). The EPR spectrum of CBC-L
2
was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu
2+
center (g
⊥
= 2.073, g
∥︀
= 2.324 G, ‖A
∥︀
‖ = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g
⊥
= 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO
C
mutants PP319 and PP359 regardless of the copper concentration in the culture medium.
RpoS (Sigma-S) Controls Expression of
rsmA
, a Global Regulator of Secondary Metabolites, Harpin, and Extracellular Proteins in
Erwinia carotovora
Authors: Mukherjee Asita , Cui Yaya , Ma Weilei , Liu Yang , Ishihama Akira , Eisenstark Abraham , Chatterjee Arun K. 
Published: December 2019

ABSTRACT

RpoS (sigma-S or sigma-38) controls a large array of genes that are expressed during stationary phase and under various stress conditions in
Escherichia coli
and other bacteria. We document here that plant pathogenic and epiphytic
Erwinia
species, such as
E. amylovora
;
E. carotovora
subsp.
atroseptica
,
betavasculorum
, and
carotovora
;
E. chrysanthemi
;
E. herbicola
;
E. rhapontici
; and
E. stewartii
, possess
rpoS
genes and produce the alternate sigma factor. We show that
rpoS
transcription in
E. carotovora
subsp.
carotovora
is driven from a major promoter which resides within the
nlpD
gene located upstream of
rpoS
as in
E. coli
. RpoS
−
E. carotovora
subsp.
carotovoa
strain AC5061, constructed by marker exchange, is more sensitive to hydrogen peroxide, carbon starvation, and acidic pH than its RpoS
+
parent strain, AC5006. The basal levels of extracellular pectate lyase, polygalacturonase, and cellulase as well as those of transcripts of
E. carotovora
subsp.
carotovora hrpN
(

hrpN
Ecc

), the gene for the elicitor of the hypersensitive reaction, are higher in the RpoS
−
strain than in the RpoS
+
parent. Likewise, compared to AC5006, AC5061 causes more extensive maceration of celery petioles. Our findings with the RpoS
−
mutant and strains carrying multiple copies
rpoS
+
DNA reveal that
rpoS
positively controls
rsmA
expression. We also present evidence that supports the hypothesis that the RpoS effect on extracellular enzyme levels,

hrpN
Ecc

expression, and virulence manifests itself by the modulation of
rsmA
expression.
Tca1, the Retrotransposon-Like Element of
Candida albicans
, Is a Degenerate and Inactive Element
Authors: Chen Jiang-ye , Wang Qin , Fu Zheng , Zhou Song , Fonzi William A. 
Published: December 2019

ABSTRACT

Candida albicans
is an asexual fungus and as such must rely on mechanisms other than sexual recombination to generate genetic diversity. Retrotransposons are ubiquitous genetic elements known to generate multiple types of genomic alterations. We have further investigated the nature of the retrotransposon-like element Tca1 in
C. albicans
. Tca1 is present at two loci in strain SC5314. Both loci have now been cloned, and one element was sequenced in its entirety. This element was flanked by α elements, or long terminal repeats (LTRs), and contained an intervening region of 5,614 bp. The intervening region was highly degenerate and contained no extended open reading frames, indicating that Tca1 is not a functional element. Partial sequence determination demonstrated that the elements from the two loci were nearly identical. Genetic manipulation of the elements showed that both loci were heterozygous for Tca1, that both were transcriptionally active, and that deletion of both had no effect on growth rate or germ tube formation. Thus, it is unclear why this nonfunctional, highly degenerate element has been maintained in many clinical isolates.
Sequence Analysis of the GntII (Subsidiary) System for Gluconate Metabolism Reveals a Novel Pathway for
l
-Idonic Acid Catabolism in
Escherichia coli
Authors: Bausch Christoph , Peekhaus Norbert , Utz Cristina , Blais Tessa , Murray Elizabeth , Lowary Todd , Conway Tyrrell 
Published: December 2019

ABSTRACT

The presence of two systems in
Escherichia coli
for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of
l
-idonic acid in which
d
-gluconate is an intermediate. The genes have been named accordingly: the
idnK
gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the
idnD-idnO-idnT-idnR
operon, which encodes
l
-idonate 5-dehydrogenase, 5-keto-
d
-gluconate 5-reductase, an
l
-idonate transporter, and an
l
-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of
l
-idonate; IdnD catalyzes a reversible oxidation of
l
-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form
d
-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of
d
-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with
l
-idonate or 5-ketogluconate serving as the true inducer of the pathway. The
l
-idonate 5-dehydrogenase and 5-keto-
d
-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of
l
-idonate and
d
-gluconate via the intermediate 5-ketogluconate.
E. coli
K-12 strains are able to utilize
l
-idonate as the sole carbon and energy source, and as predicted, the ability of
idnD
,
idnK
,
idnR
, and
edd
mutants to grow on
l
-idonate is altered.
Diversity of Cytochrome
bc
Complexes: Example of the Rieske Protein in Green Sulfur Bacteria
Authors: Brugna Myriam , Albouy Delphine , Nitschke Wolfgang 
Published: December 2019

ABSTRACT

The Rieske 2Fe2S cluster of
Chlorobium limicola
forma
thiosulfatophilum
strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone. A redox midpoint potential (

E
m

) of +160 mV in the pH range of 6 to 7.7 and a decreasing

E
m

(−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its

E
m

-versus-pH curve, in green sulfur bacteria are discussed.
Cofactor Engineering: a Novel Approach to Metabolic Engineering in
Lactococcus lactis
by Controlled Expression of NADH Oxidase
Authors: Lopez de Felipe Felix , Kleerebezem Michiel , de Vos Willem M. , Hugenholtz Jeroen 
Published: December 2019

ABSTRACT

NADH oxidase-overproducing
Lactococcus lactis
strains were constructed by cloning the
Streptococcus mutans nox-2
gene, which encodes the H
2
O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the
L. lactis nisA
promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD
+
ratios in the cells.
A Transcriptional Activator, Homologous to the
Bacillus subtilis
PurR Repressor, Is Required for Expression of Purine Biosynthetic Genes in
Lactococcus lactis
Authors: Kilstrup Mogens , Martinussen Jan 
Published: December 2019

ABSTRACT

A
purR
::pGh9:IS
S1
mutant of
Lactococcus lactis
was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxotrophs. After determination of the nucleotide sequence and deduction of the
purR
reading frame, the PurR product was found to be highly similar to the
purR
-encoded repressor from
Bacillus subtilis
. The wild-type
purR
gene complemented the purine auxotrophy of a
purR
::IS
S1
mutant, and it was shown that the
purR
::IS
S1
mutation lowered the level of transcription from the purine-regulated
L. lactis purD
promoter. In a parallel study on the regulation of
purC
and
purD
expression in
L. lactis
(M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jørgensen, M. Madsen, and D. Nilsson, J. Bacteriol. 180:3900–3906, 1998), we identified regions (PurBox sequences: AWWWCCGAACWWT) upstream of the promoters with a central G residue at exactly position −76 relative to the transcriptional start site. The PurBox sequences were found to be required for high-level promoter activity and purine regulation. We identified a PurBox sequence overlapping the −35 region of the
L. lactis purR
promoter and found, by studies of a
purR-lacLM
fusion plasmid, that
purR
is autoregulated. Because of the high degree of similarity of the PurR proteins from
B. subtilis
and
L. lactis
, we looked for PurBox sequences in the promoter regions of the PurR-regulated genes in
B. subtilis
and identified a perfectly matching PurBox sequence in the
purA
promoter region and slightly degenerate PurBox-like sequences in the promoter regions for the
pur
operon and the
purR
gene. Interestingly, the PurBox in the
pur
operon of
B. subtilis
is located almost identically, with respect to the promoter, to the PurBox sequences located in front of
purC
and
purD
in
L. lactis
. We present a hypothesis to explain how an ancestral PurR protein in
B. subtilis
could have evolved from an activator of the
pur
operon into a repressor which regulates transcription initiation from the same
pur
promoter by using the same PurR binding site and a similar response toward its effectors.
Functional Expression in
Lactobacillus plantarum
of
xylP
Encoding the Isoprimeverose Transporter of
Lactobacillus pentosus
Authors: Chaillou Stéphane , Postma Pieter W. , Pouwels Peter H. 
Published: December 2019

ABSTRACT

The
xylP
gene of
Lactobacillus pentosus
, the first gene of the
xylPQR
operon, was recently found to be involved in isoprimeverose metabolism. By expression of
xylP
on a multicopy plasmid in
Lactobacillus plantarum
80, a strain which lacks active isoprimeverose and
d
-xylose transport activities, it was shown that
xylP
encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in
L. plantarum
80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of
d
-xylose.
Investigation of theStreptomyces clavuligerusCephamycin C Gene Cluster and Its Regulation by the CcaR Protein
Authors: Alexander Dylan C. , Jensen Susan E. 
Published: December 2019

ABSTRACTAs part of a search for transcriptional regulatory genes, sequence analysis of several previously unsequenced gaps in the cephamycin biosynthetic cluster has revealed the presence inStreptomyces clavuligerusof seven genes not previously described. These include genes encoding an apparent penicillin binding protein and a transport or efflux protein, as well as the CmcI and CmcJ proteins, which catalyze late reactions in the cephamycin biosynthetic pathway. In addition, we discovered a gene, designatedpcd, which displays significant homology to genes encoding semialdehyde dehydrogenases and may represent the gene encoding the long-sought-after dehydrogenase involved in the conversion of lysine to α-aminoadipate. Finally, two genes,sclUandrhsA, with no obvious function in cephamycin biosynthesis may define the end of the cluster. The previously described CcaR protein displays homology to a number ofStreptomycespathway-specific transcriptional activators. TheccaRgene was shown to be essential for the biosynthesis of cephamycin, clavulanic acid, and non-clavulanic acid clavams. Complementation of a deletion mutant lackingccaRand the adjacentorf11andblpgenes showed that onlyccaRwas essential for the biosynthesis of cephamycin, clavulanic acid, and clavams and that mutations inorf11orblphad no discernible effects. The lack of cephamycin production inccaRmutants was directly attributable to the absence of biosynthetic enzymes responsible for the early and middle steps of the cephamycin biosynthetic pathway. Complementation of theccaRdeletion mutant resulted in the return of these biosynthetic enzymes and the restoration of cephamycin production.
Parallel and Divergent Genotypic Evolution in Experimental Populations of
Ralstonia
sp
Authors: Nakatsu Cindy H. , Korona Ryszard , Lenski Richard E. , de Bruijn Frans J. , Marsh Terence L. , Forney Larry J. 
Published: December 2019

ABSTRACT

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from
Ralstonia
sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the
tfdA
gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2.4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.
Chromosomal Integration, Tandem Amplification, and Deamplification in
Pseudomonas putida
F1 of a 105-Kilobase Genetic Element Containing the Chlorocatechol Degradative Genes from
Pseudomonas
sp. Strain B13
Authors: Ravatn Roald , Studer Sonja , Springael Dirk , Zehnder Alexander J. B. , van der Meer Jan Roelof 
Published: December 2019

ABSTRACT

Analysis of chlorobenzene-degrading transconjugants of
Pseudomonas putida
F1 which had acquired the genes for chlorocatechol degradation (
clc
) from
Pseudomonas
sp. strain B13 revealed that the
clc
gene cluster was present on a 105-kb amplifiable genetic element (named the
clc
element). In one such transconjugant,
P. putida
RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb
clc
element occurred in two different loci, and the target sites were located within the 3′ end of glycine tRNA structural genes. Tandem amplification of the
clc
element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the
clc
element. Two nonadjacent copies of the
clc
element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate. This result suggests that the presence of multiple copies of the
clc
gene cluster was a prerequisite for the growth of
P. putida
RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene.
Molecular Cloning and Characterization of
cgs
, the
Brucella abortus
Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of
Rhizobium meliloti ndvB
and
Agrobacterium tumefaciens chvB
Mutants
Authors: Iñón de Iannino Nora , Briones Gabriel , Tolmasky Marcelo , Ugalde Rodolfo A. 
Published: December 2019

ABSTRACT

The animal pathogen
Brucella abortus
contains a gene,
cgs
, that complemented a
Rhizobium meliloti
nodule development (
ndvB
) mutant and an
Agrobacterium tumefaciens
chromosomal virulence (
chvB
) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in
A. tumefaciens
, and nitrogen fixation in
R. meliloti
; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in
B. abortus
of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to
R. meliloti
NdvB. Four regions of the
B. abortus
protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with
R. meliloti
NdvB. Tn
3
-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the
Brucella
protein, although highly conserved in
Rhizobium
, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as
B. abortus
cyclic β(1-2) glucan synthetase was done by the construction of a
B. abortus
Tn
3
-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in
Brucella
infection.