bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Regulatory Mutant of
Hansenula polymorpha
Exhibiting Methanol Utilization Metabolism and Peroxisome Proliferation in Glucose
Authors: Parpinello Giuseppinia , Berardi Enrico , Strabbioli Rosanna 
Published: December 2019

ABSTRACT

Mutant LGM-128 of
Hansenula polymorpha
harbors the recessive mutation
glr2-1
which confers a complex pleiotropic phenotype, the major feature of which is the metabolically unnecessary induction of methanol utilization metabolism (C
1
metabolism) during growth on glucose, whether or not methanol is in the medium. Therefore, in this mutant, peroxisomes are formed and proliferate upon cultivation in glucose-containing media. In these media, LGM-128 shows induction levels of C
1
metabolism that are similar to those observed in methanol-containing media. This indicates that
GLR2
controls the repression-derepression process stimulated by glucose and that the induction process triggered by methanol plays only a minor role in activating C
1
metabolism. Cultivating LGM-128 in methanol and then transferring it to glucose media revealed that active degradative processes occur, leading to the disappearance of C
1
metabolism. This observation suggests that, although stimulated by glucose, the two processes are controlled by elements which are, at least in part, distinct. Finally,
glr2-1
does not affect ethanol repression, suggesting that in
H. polymorpha
the two repressing circuits are separated.
Phylogeny of Dissimilatory Sulfite Reductases Supports an Early Origin of Sulfate Respiration
Authors: Wagner Michael , Roger Andrew J. , Flax Jodi L. , Brusseau Gregory A. , Stahl David A. 
Published: December 2019

ABSTRACT

Microorganisms that use sulfate as a terminal electron acceptor for anaerobic respiration play a central role in the global sulfur cycle. Here, we report the results of comparative sequence analysis of dissimilatory sulfite reductase (DSR) genes from closely and distantly related sulfate-reducing organisms to infer the evolutionary history of DSR. A 1.9-kb DNA region encoding most of the α and β subunits of DSR could be recovered only from organisms capable of dissimilatory sulfate reduction with a PCR primer set targeting highly conserved regions in these genes. All DNA sequences obtained were highly similar to one another (49 to 89% identity), and their inferred evolutionary relationships were nearly identical to those inferred on the basis of 16S rRNA. We conclude that the high similarity of bacterial and archaeal DSRs reflects their common origin from a conserved DSR. This ancestral DSR was either present before the split between the domains
Bacteria
,
Archaea
, and
Eucarya
or laterally transferred between
Bacteria
and
Archaea
soon after domain divergence. Thus, if the physiological role of the DSR was constant over time, then early ancestors of
Bacteria
and
Archaea
already possessed a key enzyme of sulfate and sulfite respiration.
A Tn
7
-Like Transposon Is Present in the
glmUS
Region of the Obligately Chemoautolithotrophic Bacterium
Thiobacillus ferrooxidans
Authors: Oppon Joseph C. , Sarnovsky Robert J. , Craig Nancy L. , Rawlings Douglas E. 
Published: December 2019

ABSTRACT

The region downstream of the
Thiobacillus ferrooxidans
ATCC 33020
atp
operon was examined, and the genes encoding
N
-acetylglucosamine-1-uridyltransferase (
glmU
) and glucosamine synthetase (
glmS
) were found. This
atpEFHAGDC-glmUS
gene order is identical to that of
Escherichia coli
. The
T. ferrooxidans glmS
gene was shown to complement
E. coli glmS
mutants for growth on minimal medium lacking glucosamine. A Tn
7
-like transposon, Tn
5468
, was found inserted into the region immediately downstream of the
glmS
gene in a manner similar to the site-specific insertion of transposon Tn
7
within the termination region of the
E. coli glmS
gene. Tn
5468
was sequenced, and Tn
7
-like terminal repeat sequences as well as several open reading frames which are related to the Tn
7
transposition genes
tnsA
,
tnsB
,
tnsC
, and
tnsD
were found. Tn
5468
is the closest relative of Tn
7
to have been characterized to date. Southern blot hybridization indicated that a similar or identical transposon was present in three
T. ferrooxidans
strains isolated from different parts of the world but not in two
Thiobacillus thiooxidans
strains or a
Leptospirillum ferrooxidans
strain. Since
T. ferrooxidans
is an obligately acidophilic autotroph and
E. coli
is a heterotroph, ancestors of the Tn
7
-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches.
In Vivo Analysis of Sequence Requirements for Processing and Degradation of the Colicin A Lysis Protein Signal Peptide
Authors: Howard S. Peter , Lindsay Lisa 
Published: December 2019

ABSTRACT
The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide. It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures. We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal). In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine. In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide. A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis. The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells.
Multidomain Structure and Cellulosomal Localization of the
Clostridium thermocellum
Cellobiohydrolase CbhA
Authors: Zverlov Vladimir V. , Velikodvorskaya Galina V. , Schwarz Wolfgang H. , Bronnenmeier Karin , Kellermann Josef , Staudenbauer Walter L. 
Published: December 2019

ABSTRACT

The nucleotide sequence of the
Clostridium thermocellum
F7
cbhA
gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.
Transcriptional Regulation ofStreptomyces coelicolorPathway-Specific Antibiotic Regulators by theabsAandabsBLoci
Authors: Aceti David J. , Champness Wendy C. 
Published: December 2019

ABSTRACTThe four antibiotics produced byStreptomyces coelicolorare all affected by mutations in theabsAandabsBloci. TheabsAlocus encodes a putative two-component signal transduction system, and theabsBlocus encodes a homolog ofEscherichia coliRNase III. We assessed whether these loci control synthesis of the antibiotics actinorhodin and undecylprodigiosin by regulating transcript abundance from the biosynthetic and regulatory genes specific for each antibiotic. Strains that were Abs−(for antibiotic synthesis deficient) due to mutations inabsAorabsBwere examined. In the Abs−absAmutant strain, transcripts for the actinorhodin biosynthetic genesactVI-ORF1 andactI, and for the pathway-specific regulatory geneactII-ORF4, were substantially lower in abundance than in the parent strain. The level of the transcript for the undecylprodigiosin pathway-specific regulatory generedDwas similarly reduced in this mutant. Additionally, a strain that exhibits precocious hyperproduction of antibiotics (Pha phenotype) due to disruption of theabsAlocus contained elevated levels of theactVI-ORF1,actII-ORF4, andredDtranscripts. In theabsBmutant strain,actVI-ORF1,actI,actII-ORF4, andredDtranscript levels were also substantially lower than in the parent strain. These results establish that theabsgenes affect production of antibiotics through regulation of expression of the antibiotic-specific regulatory genes inS. coelicolor.
Different Modes of Diaminopimelate Synthesis and Their Role in Cell Wall Integrity: a Study with
Corynebacterium glutamicum
Authors: Wehrmann Axel , Phillipp Bodo , Sahm Hermann , Eggeling Lothar 
Published: December 2019

ABSTRACT

In eubacteria, there are three slightly different pathways for the synthesis of
m
-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is
Corynebacterium glutamicum
, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone
dapD
and prove by enzyme experiments that this gene encodes the succinylase (
M
r
= 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation,
dapD
, as well as
dapE
encoding the desuccinylase, was inactivated, thereby forcing
C. glutamicum
to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.
Regulation of
agr
-Dependent Virulence Genes in
Staphylococcus aureus
by RNAIII from Coagulase-Negative Staphylococci
Authors: Tegmark Karin , Morfeldt Eva , Arvidson Staffan 
Published: December 2019

ABSTRACT

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in
Staphylococcus aureus
are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including
hla
(alpha-toxin),
saeB
(enterotoxin B),
tst
(toxic shock syndrome toxin 1), and
ssp
(serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like
spa
(protein A) and
fnb
(fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e.,
Staphylococcus epidermidis
,
Staphylococcus simulans
, and
Staphylococcus warneri
. RNAIII from these CoNS turned out to be very similar to that of
S. aureus
and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5′ half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient
S. aureus
mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of
S. aureus
RNAIII, suggesting that these regulatory functions are independent. By creating
S. epidermidis-S. aureus
RNAIII hybrids, we could also show that both the 5′ and 3′ halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in
S. aureus.
Cloning and Comparison of
fliC
Genes and Identification of Glycosylation in the Flagellin of
Pseudomonas aeruginosa
a-Type Strains
Authors: Brimer Cynthia D. , Montie T. C. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several
Pseudomonas aeruginosa
a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.
The Glucose Kinase of
Bacillus subtilis
Authors: Skarlatos Pierre , Dahl Michael K. 
Published: December 2019

ABSTRACT

The open reading frame
yqgR
(now termed
glcK
), which had been sequenced as part of the genome project, encodes a glucose kinase of
Bacillus subtilis
. A 1.1-kb DNA fragment containing
glcK
complemented an
Escherichia coli
strain deficient in glucose kinase activity. Insertional mutagenesis of
glcK
resulted in a complete inactivation of glucose kinase activity in crude protein extracts, indicating that
B. subtilis
contains one major glucose kinase. The
glcK
gene encodes a 321-residue protein with a molecular mass of 33.5 kDa. The glucose kinase was overexpressed as a fusion protein to a six-His affinity tag and purified to homogeneity. The enzyme had

K
m

values for ATP and glucose of 0.77 and 0.24 mM, respectively, and a
V
max
of 93 μmol min
−1
mg
−1
. A
B. subtilis
strain deficient for glucose kinase grew at the same rate on different carbon sources tested, including disaccharides such as maltose, trehalose, and sucrose.
Mutagenesis and Expression of
amo
, Which Codes for Ammonia Monooxygenase in
Nitrosomonas europaea
Authors: Hommes Norman G. , Sayavedra-Soto Luis A. , Arp Daniel J. 
Published: December 2019

ABSTRACT

Nitrosomonas europaea
has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of
amoA
were obtained, and they were found to differ by one nucleotide. To determine if both copies of
amoA
were functional, insertional mutagenesis was performed to inactivate either copy of
amoA
alone. A DNA cassette containing the
lacZ
and
kan
genes inserted into
amoA
was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of
amoA
. Mutations were obtained in both copies of
amoA
. Either copy of
amoA
was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of
amoA
, but not the other, resulted in slower growth. Measurements of ammonia-dependent O
2
consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [
14
C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.
Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(
RS
)-2-(2,4-Dichlorophenoxy)propanoic acid] by
Sphingomonas herbicidovorans
MH
Authors: Zipper Christian , Bunk Monika , Zehnder Alexander J. B. , Kohler Hans-Peter E. 
Published: December 2019

ABSTRACT

Sphingomonas herbicidovorans
MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(
RS
)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (
S
) enantiomer over the (
R
) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(
RS
)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (

K
t

) of 108, 93, and 117 μM and maximal velocities (
V
max
) of 19, 10, and 21 nmol min
−1
mg of protein
−1
for (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D, respectively. Transport of (
R
)-dichlorprop, (
S
)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor
N
,
N
′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (
R
)-dichlorprop uptake was inhibited by (
R
)-mecoprop but not by (
S
)-mecoprop, (
S
)-dichlorprop, or 2,4-D. On the other hand, the (
S
)-dichlorprop transport was inhibited by (
S
)-mecoprop but not by (
R
)-mecoprop, (
R
)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by
S. herbicidovorans
is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (
R
)-dichlorprop and (
R
)-mecoprop, another for (
S
)-dichlorprop and (
S
)-mecoprop, and a third for 2,4-D.
Identification of a Specific Chaperone for SptP, a Substrate of the Centisome 63 Type III Secretion System of
Salmonella typhimurium
Authors: Fu Yixin , Galán Jorge E. 
Published: December 2019

ABSTRACT

Salmonella typhimurium
uses of a type III protein secretion system encoded at centisome 63 of its chromosome to deliver effector molecules into the host cell. These proteins stimulate host cell responses such as reorganization of the actin cytoskeleton and activation of transcription factors. One of these effector proteins is SptP, a tyrosine phosphatase that causes disruption of the host cell actin cytoskeleton. A characteristic feature of many substrates of type III secretion systems is their association with specific cytoplasmic chaperones which appears to be required for secretion and/or translocation of these proteins into the host cell. We report here the identification of SicP, a 13-kDa acidic polypeptide that is encoded immediately upstream of
sptP
. A loss-of-function mutation in
sicP
resulted in drastically reduced levels of SptP but did not affect
sptP
expression, indicating that SicP exerts its effect posttranscriptionally. Pulse-chase experiments demonstrated that the loss of SicP leads to increased degradation of SptP. In addition, we show that SicP binds to SptP directly and that the binding site is located between residues 15 and 100 of the tyrosine phosphatase. Taken together, these results indicate that SicP acts as a specific chaperone for SptP.
Cloning, Sequencing, and Phenotypic Characterization of the
rpoS
Gene from
Pseudomonas putida
KT2440
Authors: Ramos-González María Isabel , Molin Søren 
Published: December 2019

ABSTRACT

A gene homologous to the
rpoS
gene of
Escherichia coli
was cloned from a
Pseudomonas putida
KT2440 gene bank by complementation of the
rpoS
-deficient strain
E. coli
ZK918. The
rpoS
gene of
P. putida
complemented the acid sensitivity and catalase deficiency of the
rpoS
mutant of
E. coli
and stimulated expression of the RpoS-controlled promoter,
bolAp
1
. The gene was sequenced and found to be highly similar to the
rpoS
genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of the
nlpD
gene was found upstream to the
rpoS
gene. A transcriptional fusion of the promoter of the
P. putida rpoS
gene to the
luxAB
genes from
Vibrio harveyi
was constructed and used as an inactivated allele of
rpoS
for gene replacement of the wild-type copy in the chromosome of
P. putida
. The resultant
rpoS
mutant of
P. putida
, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with
m
-methylbenzoate was also reduced in the mutant strain
P. putida
C1R1. The RpoS protein of
P. putida
controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.