bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel Cellulosomal Scaffoldin from
Acetivibrio cellulolyticus
That Contains a Family 9 Glycosyl Hydrolase
Authors: Ding Shi-You , Bayer Edward A. , Steiner David , Shoham Yuval , Lamed Raphael 
Published: December 2019

ABSTRACT

A novel cellulosomal scaffoldin gene, termed
cipV
, was identified and sequenced from the mesophilic cellulolytic anaerobe
Acetivibrio cellulolyticus
. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of
Clostridium thermocellum
. The
cipV
gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted “recognition codes” differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of
C. thermocellum
and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from
C. thermocellum
as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.
Influence of
S
-Adenosylmethionine Pool Size on Spontaneous Mutation, Dam Methylation, and Cell Growth of
Escherichia coli
Authors: Posnick Lauren M. , Samson Leona D. 
Published: December 2019

ABSTRACT

Escherichia coli
strains that are deficient in the Ada and Ogt DNA repair methyltransferases display an elevated spontaneous G:C-to-A:T transition mutation rate, and this increase has been attributed to mutagenic
O
6
-alkylguanine lesions being formed via the alkylation of DNA by endogenous metabolites. Here we test the frequently cited hypothesis that
S
-adenosylmethionine (SAM) can act as a weak alkylating agent in vivo and that it contributes to endogenous DNA alkylation. By regulating the expression of the rat liver SAM synthetase and the bacteriophage T3 SAM hydrolase proteins in
E. coli
, a 100-fold range of SAM levels could be achieved. However, neither increasing nor decreasing SAM levels significantly affected spontaneous mutation rates, leading us to conclude that SAM is not a major contributor to the endogenous formation of
O
6
-methylguanine lesions in
E. coli
.
Identification and Disruption of
lisRK
, a Genetic Locus Encoding a Two-Component Signal Transduction System Involved in Stress Tolerance and Virulence in
Listeria monocytogenes
Authors: Cotter Paul D. , Emerson Nathan , Gahan Cormac G. M. , Hill Colin 
Published: December 2019

ABSTRACT

lisRK
encodes a two-component regulatory system in the food pathogen
Listeria monocytogenes
LO28. Following identification of the operon in an acid-tolerant Tn
917
mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.
Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of
Streptococcus salivarius
Authors: Plamondon Pascale , Brochu Denis , Thomas Suzanne , Fradette Julie , Gauthier Lucie , Vaillancourt Katy , Buckley Nicole , Frenette Michel , Vadeboncoeur Christian 
Published: December 2019

ABSTRACT

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His
15
) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser
46
) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from
Streptococcus salivarius
ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in
ptsH
(the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His
15
by EI nor the phosphorylation at Ser
46
by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.
IS
1397
Is Active for Transposition into the Chromosome of
Escherichia coli
K-12 and Inserts Specifically into Palindromic Units of Bacterial Interspersed Mosaic Elements
Authors: Clément Jean-Marie , Wilde Caroline , Bachellier Sophie , Lambert Patricia , Hofnung Maurice 
Published: December 2019

ABSTRACT

We demonstrate that IS
1397
, a putative mobile genetic element discovered in natural isolates of
Escherichia coli
, is active for transposition into the chromosome of
E. coli
K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to
E. coli
and
Salmonella
. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.
Molecular Cloning and Characterization of the
Helicobacter pylori fliD
Gene, an Essential Factor in Flagellar Structure and Motility
Authors: Seong Kim Jang , Hoon Chang Ji , Il Chung Soo , Sun Yum Jung 
Published: December 2019

ABSTRACT

Helicobacter pylori
colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of
H. pylori
. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The
fliD
operon of
H. pylori
consists of
flaG
,
fliD
, and
fliS
genes, in the order stated, under the control of a ς
28
-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the
fliD
gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the
fliD
gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the
fliD
mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The
fliD
mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic
fliD
mutant of
H. pylori
SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that
H. pylori
FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.
TPW22, a Lactococcal Temperate Phage with a Site-Specific Integrase Closely Related to
Streptococcus thermophilus
Phage Integrases
Authors: Petersen Anne , Josephsen Jytte , Johnsen Mads G. 
Published: December 2019

ABSTRACT

The temperate phage TPW22, induced from
Lactococcus lactis
subsp.
cremoris
W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on
L. lactis
subsp.
cremoris
3107, which could also be lysogenized by site-specific integration. The attachment site (
attP
), 5′-TAAGGCGACGGTCG-3′, of phage TPW22 was present on a 7.5-kb
Eco
RI fragment, a 3.4-kb
Eco
RI-
Hin
dIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (
int
). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing
attP
and
int
was constructed, and integration in
L. lactis
subsp.
cremoris
MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages φLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the φLC3 lactococcal integrase with known
Streptococcus thermophilus
integrases.
Functional Regions of the
Bacillus subtilis
Spore Coat Morphogenetic Protein CotE
Authors: Bauer Tamara , Little Shawn , Stöver Axel G. , Driks Adam 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat’s outer layer. We have used a series of mutant alleles of
cotE
to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.
A Mutation in the 3-Phosphoglycerate Kinase Gene Allows Anaerobic Growth of
Bacillus subtilis
in the Absence of ResE Kinase
Authors: Nakano Michiko M. , Zhu Yi , Haga Koki , Yoshikawa Hirofumi , Sonenshein Abraham L. , Zuber Peter 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to
pgk
, the gene encoding 3-phosphoglycerate kinase, failed to suppress a
resD
mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The
pgk-1
mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1,3-diphosphoglycerate, is responsible for the observed effect of the
pgk-1
mutation on anaerobiosis of
resE
mutant cells.
Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from
Prevotella intermedia
Authors: Chen Xiaochi , Ansai Toshihiro , Awano Shuji , Iida Toshiya , Barik Sailen , Takehara Tadamichi 
Published: December 2019

ABSTRACT

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from
Prevotella intermedia
ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an
M
r
of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of
Morganella morganii
, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.
Roles of Three Histidine Kinase Genes in Hyphal Development and Virulence of the Pathogenic Fungus
Candida albicans
Authors: Yamada-Okabe Toshiko , Mio Toshiyuki , Ono Naomi , Kashima Yuji , Matsui Mitsuaki , Arisawa Mikio , Yamada-Okabe Hisafumi 
Published: December 2019

ABSTRACT

The pathogenic fungus
Candida albicans
harbors three histidine kinase genes called
CaSLN1
,
CaNIK1
, and
CaHK1
. The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of
C. albicans
in a mouse systemic candidiasis model. The effects of the disruption on hyphal formation and virulence were most severe in the
cahk1
Δ null mutants. Although the double disruption of
CaSLN1
and
CaNIK1
was impossible, further deletion of
CaSLN1
or
CaNIK1
in the
cahk1
Δ null mutants partially restored the serum-induced hypha-forming ability and virulence. When incubated with radiolabelled ATP, the recombinant CaSln1 and CaNik1 proteins, which contained their own kinase and response regulator domains, were autophosphorylated, whereas CaHk1p was not. These results imply that in
C. albicans
,
CaSLN1
and
CaNIK1
function upstream of
CaHK1
but are in distinct signal transmission pathways.
A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Sakamoto Toshio , Inoue-Sakamoto Kaori , Bryant Donald A. 
Published: December 2019

ABSTRACT

The
nrtP
and
narB
genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium
Synechococcus
sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An
nrtP
mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The
nrtP
mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A
narB
mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the
narB
mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the
nrtP
and
narB
genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the
nrtP
and
narB
genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in
Synechococcus
sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.
Regulation of
adhE
(Encoding Ethanol Oxidoreductase) by the Fis Protein in
Escherichia coli
Authors: Membrillo-Hernández Jorge , Kwon Ohsuk , De Wulf Peter , Finkel Steven E. , Lin E. C. C. 
Published: December 2019

ABSTRACT

The
adhE
gene of
Escherichia coli
encodes a multifunctional ethanol oxidoreductase whose expression is 10-fold higher under anaerobic than aerobic conditions. Transcription of the gene is under the negative control of the Cra (catabolite repressor-activator) protein, whereas translation of the
adhE
mRNA requires processing by RNase III. In this report, we show that the expression of
adhE
also depends on the Fis (factor for inversion stimulation) protein. A strain bearing a
fis
::
kan
null allele failed to grow anaerobically on glucose solely because of inadequate
adhE
transcription. However,
fis
expression itself is not under redox control. Sequence inspection of the
adhE
promoter revealed three potential Fis binding sites. Electrophoretic mobility shift analysis, using purified Fis protein and
adhE
promoter DNA, showed three different complexes.
Genetic Analysis of a Chromosomal Region Containing Genes Required for Assimilation of Allantoin Nitrogen and Linked Glyoxylate Metabolism in
Escherichia coli
Authors: Cusa Eva , Obradors Nuria , Baldomà Laura , Badía Josefa , Aguilar Juan 
Published: December 2019

ABSTRACT

Growth experiments with
Escherichia coli
have shown that this organism is able to use allantoin as a sole nitrogen source but not as a sole carbon source. Nitrogen assimilation from this compound was possible only under anaerobic conditions, in which all the enzyme activities involved in allantoin metabolism were detected. Of the nine genes encoding proteins required for allantoin degradation, only the one encoding glyoxylate carboligase (
gcl
), the first enzyme of the pathway leading to glycerate, had been identified and mapped at centisome 12 on the chromosome map. Phenotypic complementation of mutations in the other two genes of the glycerate pathway, encoding tartronic semialdehyde reductase (
glxR
) and glycerate kinase (
glxK
), allowed us to clone and map them closely linked to
gcl
. Complete sequencing of a 15.8-kb fragment encompassing these genes defined a regulon with 12 open reading frames (ORFs). Due to the high similarity of the products of two of these ORFs with yeast allantoinase and yeast allantoate amidohydrolase, a systematic analysis of the gene cluster was undertaken to identify genes involved in allantoin utilization. A BLASTP search predicted four of the genes that we sequenced to encode allantoinase (
allB
), allantoate amidohydrolase (
allC
), ureidoglycolate hydrolase (
allA
), and ureidoglycolate dehydrogenase (
allD
). The products of these genes were overexpressed and shown to have the predicted corresponding enzyme activities. Transcriptional fusions to
lacZ
permitted the identification of three functional promoters corresponding to three transcriptional units for the structural genes and another promoter for the regulatory gene
allR
. Deletion of this regulatory gene led to constitutive expression of the regulon, indicating a negatively acting function.
Vir Proteins Stabilize VirB5 and Mediate Its Association with the T Pilus of
Agrobacterium tumefaciens
Authors: Schmidt-Eisenlohr Heike , Domke Natalie , Angerer Christina , Wanner Gerhard , Zambryski Patricia C. , Baron Christian 
Published: December 2019

ABSTRACT

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from
Agrobacterium tumefaciens
, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711–2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes
virB1
,
virB2
,
virB5
, and
virB6
blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.