bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the
fnrL
Gene in Photosystem Gene Expression and Photosynthetic Growth of
Rhodobacter sphaeroides
2.4.1
Authors: Zeilstra-Ryalls Jill H. , Kaplan Samuel 
Published: December 2019

ABSTRACT

Anoxygenic photosynthetic growth of
Rhodobacter sphaeroides
2.4.1 requires a functional
fnrL
gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the
puc
operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in
puc
operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of
prr
or
ppsR
can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional
fnrL
gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode.
Xanthomonas campestris
pv. campestris
gum
Mutants: Effects on Xanthan Biosynthesis and Plant Virulence
Authors: Katzen Federico , Ferreiro Diego U. , Oddo Cristian G. , Ielmini M. Verónica , Becker Anke , Pühler Alfred , Ielpi Luis 
Published: December 2019

ABSTRACT

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium
Xanthomonas campestris
pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated
xpsI
or
gum
has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of
X. campestris gum
mutants, we report experimental data for assigning functions to the products of the
gum
genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain
gum
and non-
gum
mutations. In addition, we provide evidence that the C-terminal domain of the
gumD
gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of
X. campestris
against the plant.
Characterization of
dnaC2
and
dnaC28
Mutants by Flow Cytometry
Authors: Withers Helen L. , Bernander Rolf 
Published: December 2019

ABSTRACT

Escherichia coli
strains containing thermosensitive
dnaC
alleles were studied by flow cytometry. Strains containing either the
dnaC2
or
dnaC28
allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of
dnaC
mutants for cell cycle studies is discussed.
Cloning and Characterization of the
Pseudomonas aeruginosa zwf
Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat)
Authors: Ma Ju-Fang , Hager Paul W. , Howell Michael L. , Phibbs Paul V. , Hassett Daniel J. 
Published: December 2019

ABSTRACT

In this study, we cloned the
Pseudomonas aeruginosa zwf
gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD
+
- or NADP
+
-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted
zwf
gene product is 490 residues, which could form a tetramer with a molecular mass of ∼220 kDa. G6PDH activity and
zwf
transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and
zwf
transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be ∼220 kDa by size exclusion chromatography. Estimated

K
m

values of purified G6PDH acting on glucose-6-phosphate, NADP
+
, and NAD
+
were 530, 57, and 333 μM, respectively. The specific activities with NAD
+
and NADP
+
were calculated to be 176 and 69 μmol/min/mg. An isogenic
zwf
mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.
Genetic Complementation and Kinetic Analyses of
Rhodobacter capsulatus ORF1696
Mutants Indicate that the ORF1696 Protein Enhances Assembly of the Light-Harvesting I Complex
Authors: Young C. S. , Reyes R. C. , Beatty J. T. 
Published: December 2019

ABSTRACT

Rhodobacter capsulatus ORF1696
mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the
ORF1696
gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of
ORF1696
resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when
trans
complemented with a plasmid expressing the
ORF1696
gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in
ORF1696
. We suggest that a
rho
-dependent transcription termination site exists between the
Mun
I and proximal
Stu
I sites of
ORF1696
. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the
ORF1696
parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.
Promoter Characterization and Constitutive Expression of the
Escherichia coli gcvR
Gene
Authors: Ghrist Angela C. , Stauffer George V. 
Published: December 2019

ABSTRACT

The
Escherichia coli
glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (
gcv
). In this study, the
gcvR
translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the
gcvR
promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a
gcvR-lacZ
fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of
gcv
, respectively. In addition, it was shown that
gcvR-lacZ
expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream
bcp
gene overlaps the
gcvR
stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of
gcvR
had no effect on the expression of a downstream
bcp-lacZ
fusion, and primer extension analysis of the
bcp
promoter region demonstrated that
bcp
has its own promoter within the
gcvR
coding sequence. These results show that
gcvR
and
bcp
do not form an operon. Furthermore, the deletion of
bcp
from the chromosome had no effect on
gcv-lacZ
expression.
Acquisition of Five High-
M
r
Penicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from
Streptococcus mitis
to
Streptococcus pneumoniae
Authors: Hakenbeck Regine , König Andrea , Kern Izabella , van der Linden Mark , Keck Wolfgang , Billot-Klein Danielle , Legrand Raymond , Schoot Bernard , Gutmann Laurent 
Published: December 2019

ABSTRACT

Penicillin-resistant isolates of
Streptococcus pneumoniae
generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of
S. pneumoniae
obtained with chromosomal donor DNA from a
Streptococcus mitis
isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the
S. mitis
donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental
S. pneumoniae
strain. The results stress the potential for the acquisition by
S. pneumoniae
of high-level β-lactam resistance by interspecies gene transfer.
Chemotactic Adaptation Is Altered by Changes in the Carboxy-Terminal Sequence Conserved among the Major Methyl-Accepting Chemoreceptors
Authors: Okumura Hisashi , Nishiyama So-ichiro , Sasaki Akie , Homma Michio , Kawagishi Ikuro 
Published: December 2019

ABSTRACT

In
Escherichia coli
and
Salmonella typhimurium
, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless
E. coli
strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.
ntn
Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in
Pseudomonas
sp. Strain TW3
Authors: James Keith D. , Williams Peter A. 
Published: December 2019

ABSTRACT

Pseudomonas
sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes,
ntnWCMAB
*, which are very similar to and in the same order as the
xyl
operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (
ntnW
), an NAD
+
-linked benzaldehyde dehydrogenase (
ntnC
), a two-gene toluene monooxygenase (
ntnMA
), and part of a benzyl alcohol dehydrogenase (
ntnB
*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding
xylWCMAB
genes. The
xylB
homolog on the TW3 genome (
ntnB
*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway.
Variation of the Ribosomal Operon 16S-23S Gene Spacer Region in Representatives of
Salmonella enterica
Subspecies
Authors: Luz Sara Pérez , Rodríguez-Valera Francisco , Lan Ruiting , Reeves Peter R. 
Published: December 2019

ABSTRACT

The 16S-23S spacer regions of two ribosomal operons (
rrnA
and
rrnE
) have been sequenced in seven representatives of the
Salmonella enterica
subspecies. Isolated nucleotide substitutions were found at the same sites as in
Escherichia coli
but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in
E. coli
, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of
rrnE
put subspecies I closer to
E. coli
K-12 than to the other
S. enterica
subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in
E. coli
, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family
Enterobacteriaceae
were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.
An AT-Rich Tract Containing an Integration Host Factor-Binding Domain and Two UP-Like Elements Enhances Transcription from the
pilEp
1
Promoter of
Neisseria gonorrhoeae
Authors: Fyfe Janet A. M. , Davies John K. 
Published: December 2019

ABSTRACT

The
pilE
gene of
Neisseria gonorrhoeae
is transcribed from a ς
70
promoter (
pilEp
1
) with an AT-rich tract extending 65 nucleotides upstream of the −35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription from
pilEp
1
in
N. gonorrhoeae
. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both
N. gonorrhoeae
and an
Escherichia coli
strain producing IHF. In addition, an UP-like element positioned between the −35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the −35 box.
A
Bacillus subtilis
Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase
Authors: Aguilar Pablo S. , Cronan John E. , de Mendoza Diego 
Published: December 2019

ABSTRACT

Bacillus subtilis
grown at 37°C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37°C are transferred to 20°C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of
B. subtilis
. This gene, called
des
, was isolated by complementation of
Escherichia coli
strains with mutations in either of two different genes of UFA synthesis. The
des
gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the
des
gene in
E. coli
resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA
cis
-5-hexadecenoic acid, indicating that the
B. subtilis des
gene product is a Δ5 acyl-lipid desaturase. The
des
gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The
des
null mutant strain grew as well as its congenic parent at 20 or 37°C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the
des
promoter directed the synthesis of a
lacZ
reporter gene showed that
des
expression is repressed at 37°C, but a shift of cultures from 37 to 20°C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.
Oxygen Regulation of the
ccoN
Gene Encoding a Component of the
cbb
3
Oxidase in
Rhodobacter sphaeroides
2.4.1
T
: Involvement of the FnrL Protein
Authors: Mouncey Nigel J. , Kaplan Samuel 
Published: December 2019

ABSTRACT

The
ccoNOQP
gene cluster of
Rhodobacter sphaeroides
2.4.1
T
encodes a
cbb
3
cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration. The β-galactosidase activity of a
ccoN
::
lacZ
transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions. Maximal
ccoN
::
lacZ
expression was observed when the oxygen concentration was lowered to 2%. In an FnrL mutant,
ccoN
::
lacZ
expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding the
cbb
3
oxidase.
Regulation of Lactose Utilization Genes in
Staphylococcus xylosus
Authors: Bassias Joannis , Brückner Reinhold 
Published: December 2019

ABSTRACT

The lactose utilization genes of
Staphylococcus xylosus
have been isolated and characterized. The system is comprised of two structural genes,
lacP
and
lacH
, encoding the lactose permease and the β-galactosidase proteins, respectively, and a regulatory gene,
lacR
, coding for an activator of the AraC/XylS family. The lactose utilization genes are divergently arranged, the
lacPH
genes being opposite to
lacR
. The
lacPH
genes are cotranscribed from one promoter in front of
lacP
, whereas
lacR
is transcribed from two promoters of different strengths. Lactose transport as well as β-galactosidase activity are inducible by the addition of lactose to the growth medium. Primer extension experiments demonstrated that regulation is achieved at the level of
lacPH
transcription initiation. Inducibility and efficient
lacPH
transcription are dependent on a functional
lacR
gene. Inactivation of
lacR
resulted in low and constitutive
lacPH
expression. Expression of
lacR
itself is practically constitutive, since transcription initiated at the major
lacR
promoter does not respond to the availability of lactose. Only the minor
lacR
promoter is lactose inducible. Apart from lactose-specific, LacR-dependent control, the
lacPH
promoter is also subject to carbon catabolite repression mediated by the catabolite control protein CcpA. When glucose is present in the growth medium,
lacPH
transcription initiation is reduced. Upon
ccpA
inactivation, repression at the
lacPH
promoter is relieved. Despite this loss of transcriptional regulation in the
ccpA
mutant strain, β-galactosidase activity is still reduced by glucose, suggesting another level of control.
Metalloadsorption by
Escherichia coli
Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB
Authors: Sousa Carolina , Kotrba Pavel , Ruml Tomas , Cebolla Angel , De Lorenzo Víctor 
Published: December 2019

ABSTRACT

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in
Escherichia coli
as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of
Saccharomyces cerevisiae
(yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB
153
-YMT and LamB
153
-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of
E. coli
cells to bind Cd
2+
15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.