bluuebunny/crossref_metadata_2025_split huggingface dataset:
Facilitation of Direct Conditional Knockout of Essential Genes in
Bacillus licheniformis
DSM13 by Comparative Genetic Analysis and Manipulation of Genetic Competence
Authors: Hoffmann Kerstin , Wollherr Antje , Larsen Michael , Rachinger Michael , Liesegang Heiko , Ehrenreich Armin , Meinhardt Friedhelm 
Published: June 2010

ABSTRACT

The genetic manageability of the biotechnologically important
Bacillus licheniformis
is hampered due to its poor transformability, whereas
Bacillus subtilis
efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing module
comQXPA
of
B. licheniformis
DSM13, was found to be inactive due to an insertion element within
comP
, the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, in
trans
resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density in
B. subtilis
), we performed an
in silico
analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of the
comQXPA
-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations.
Biosynthesis and Biodegradation of 3-Hydroxypropionate- Containing Polyesters
Authors: Andreeßen Björn , Steinbüchel Alexander 
Published: June 2010

ABSTRACT

3-Hydroxypropionate (3HP) is an important compound in the chemical industry, and the polymerized 3HP can be used as a bioplastic. In this review, we focus on polyesters consisting of 3HP monomers, including the homopolyester poly(3-hydroxypropionate) and copolyesters poly(3-hydroxybutyrate-
co
-3-hydroxypropionate), poly(3-hydroxypropionate-
co
-3-hydroxybutyrate-
co
-3-hydroxyhexanoate-
co
-3-hydroxyoctanoate), poly(4-hydroxybutyrate-
co
-3-hydroxypropionate-
co
-lactate), and poly(3-hydroxybutyrate-
co
-3-hydroxypropionate-
co
-4-hydroxybutyrate-
co
-lactate). Homopolyesters like poly(3-hydroxybutyrate) are often highly crystalline and brittle, which limits some of their applications. The incorporation of 3HP monomers reduces the glass transition temperature, the crystallinity, and also, at up to 60 to 70 mol% 3HP, the melting point of the copolymer. This review provides a survey of the synthesis and physical properties of different polyesters containing 3HP.
Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of
Cronobacter sakazakii
Authors: Kim Kyumson , Kim Kwang-Pyo , Choi Jeongjoon , Lim Jeong-A , Lee Junghyun , Hwang Sunyoung , Ryu Sangryeol 
Published: June 2010

ABSTRACT

Cronobacter sakazakii
is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of
C. sakazakii
, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The
ompX
and
ompA
deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by
C. sakazakii
. This is the first report of
C. sakazakii
virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of
C. sakazakii
.
Improved
dsrA
-Based Terminal Restriction Fragment Length Polymorphism Analysis of Sulfate-Reducing Bacteria
Authors: Santillano Daniel , Boetius Antje , Ramette Alban 
Published: June 2010

ABSTRACT

To better describe the community structure of sulfate-reducing bacteria in environmental systems, we compared several dissimilatory sulfite reductase (
dsr
) primer sets for terminal restriction fragment length polymorphism application. A new reverse primer that increased allelic diversity estimates up to 5-fold was applied to hydrocarbon seep samples to examine the relationship between guild activity and diversity.
Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures
Authors: Walker Claire A. , Gómez Beatriz L. , Mora-Montes Héctor M. , Mackenzie Kevin S. , Munro Carol A. , Brown Alistair J. P. , Gow Neil A. R. , Kibbler Christopher C. , Odds Frank C. 
Published: June 2010

ABSTRACT

The fungal pathogen
Candida albicans
produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing
l
-3,4-dihydroxyphenylalanine (
l
-DOPA) as a substrate. Expression profiling of
C. albicans
revealed very few genes significantly up- or downregulated by growth in
l
-DOPA. We were unable to determine a possible role for melanin in the virulence of
C. albicans
. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of
N
-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene
CHS2
. Melanosomes remained within the outermost cell wall layers in
chs3
Δ and
chs2
Δ
chs3
Δ mutants but were fully externalized in
chs8
Δ and
chs2
Δ
chs8
Δ mutants. All the
CHS
mutants synthesized dark pigment at equivalent rates from mixed membrane fractions
in vitro
, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes
CHT2
and
CHT3
and the chitin pathway regulator
ECM33
also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of
CHS2
, impedes externalization.
β(1-3)Glucanosyltransferase Gel4p Is Essential for Aspergillus fumigatus
Authors: Gastebois Amandine , Fontaine Thierry , Latgé Jean-Paul , Mouyna Isabelle 
Published: June 2010

ABSTRACT

The β(1-3)glucanosyltransferase
GEL
family of
Aspergillus fumigatus
contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the
GEL4
gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other β(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.
Genome Organization and Pathogenicity ofCorynebacterium diphtheriaeC7(−) and PW8 Strains
Authors: Iwaki Masaaki , Komiya Takako , Yamamoto Akihiko , Ishiwa Akiko , Nagata Noriyo , Arakawa Yoshichika , Takahashi Motohide 
Published: June 2010

ABSTRACTCorynebacterium diphtheriaeis the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(−) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(−). Another importantC. diphtheriaestrain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(−) genome. Despite this, the C7(−) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among variousC. diphtheriaestrains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.
Frameshift Mutations in a Single Novel Virulence Factor Alter theIn VivoPathogenicity ofChlamydia trachomatisfor the Female Murine Genital Tract
Authors: Sturdevant Gail L. , Kari Laszlo , Gardner Donald J. , Olivares-Zavaleta Norma , Randall Linnell B. , Whitmire William M. , Carlson John H. , Goheen Morgan M. , Selleck Elizabeth M. , Martens Craig , Caldwell Harlan D. 
Published: June 2010

ABSTRACTChlamydia trachomatisis a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model ofC. trachomatisinfection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivatedin vitroprior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiplein vitropassages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrastingin vivovirulence characteristics, the strains exhibited no differences in the results ofin vitroinfectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.
Characterization of the Transposase Encoded by IS
256
, the Prototype of a Major Family of Bacterial Insertion Sequence Elements
Authors: Hennig Susanne , Ziebuhr Wilma 
Published: June 2010

ABSTRACT

IS
256
is the founding member of the IS
256
family of insertion sequence (IS) elements. These elements encode a poorly characterized transposase, which features a conserved DDE catalytic motif and produces circular IS intermediates. Here, we characterized the IS
256
transposase as a DNA-binding protein and obtained insight into the subdomain organization and functional properties of this prototype enzyme of IS
256
family transposases. Recombinant forms of the transposase were shown to bind specifically to inverted repeats present in the IS
256
noncoding regions. A DNA-binding domain was identified in the N-terminal part of the transposase, and a mutagenesis study targeting conserved amino acid residues in this region revealed a putative helix-turn-helix structure as a key element involved in DNA binding. Furthermore, we obtained evidence to suggest that the terminal nucleotides of IS
256
are critically involved in IS circularization. Although small deletions at both ends reduced the formation of IS circles, changes at the left-hand IS
256
terminus proved to be significantly more detrimental to circle production. Taken together, the data lead us to suggest that the IS
256
transposase-mediated circularization reaction preferentially starts with a sequence-specific first-strand cleavage at the left-hand IS terminus.
Changes in the Min Oscillation Pattern before and after Cell Birth
Authors: Juarez Jennifer R. , Margolin William 
Published: June 2010

ABSTRACT

The Min system regulates the positioning of the cell division site in many bacteria. In
Escherichia coli
, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a
gfp-minD
fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.
Catabolite Repression of the TodS/TodT Two-Component System and Effector-Dependent Transphosphorylation of TodT as the Basis for Toluene Dioxygenase Catabolic Pathway Control
Authors: Busch Andreas , Lacal Jesús , Silva-Jímenez Hortencia , Krell Tino , Ramos Juan L. 
Published: June 2010

ABSTRACT

The TodS/TodT two-component system of
Pseudomonas putida
regulates the expression of the toluene dioxygenase (
tod
) operon for the metabolism of toluene, benzene, and ethylbenzene. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor and an autokinase domain separated by a receiver domain. The TodT protein is the cognate response regulator that activates transcription of the toluene dioxygenase (TOD) pathway genes at the P

todX

promoter. We report in this study that the
todST
operon is transcribed from a main promoter and that the +1 initiation point is located 31 nucleotides upstream from the A of the first ATG codon and is preceded by a −10/−35 canonical promoter. Expression from P

todS

is under catabolite control, and in cells growing with glucose, the level of expression from this promoter is reduced, which in turn translates to low levels of the TodS/TodT regulators and results in a decrease of transcription from the P

todX

promoter. Thus, the main underlying regulatory mechanisms of the
tod
structural genes are at the levels of catabolite repression control from P

todS

and transcription activation, mediated by the TodT response regulator through a regulatory cascade in which the effector enhances autophosphorylation of TodS by ATP, with subsequent transphosphorylation of TodT.
Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation inMyxococcus xanthus
Authors: Black Wesley P. , Schubot Florian D. , Li Zhuo , Yang Zhaomin 
Published: June 2010

ABSTRACTMyxococcus xanthussocial gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as adifD difEdouble mutant phenocopied thedifEsingle mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we reportin vitrophosphorylation studies with all the Dif chemosensory proteins that were expressed and purified fromEscherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylationin vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.
Identification of the Biosynthetic Gene Cluster for thePseudomonas aeruginosaAntimetabolitel-2-Amino-4-Methoxy-trans-3-Butenoic Acid
Authors: Lee Xiaoyun , Fox Áine , Sufrin Janice , Henry Hugues , Majcherczyk Paul , Haas Dieter , Reimmann Cornelia 
Published: June 2010

ABSTRACTl-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced byPseudomonas aeruginosa.Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.
Genome Sequence of a Cellulose-Producing Bacterium,
Gluconacetobacter hansenii
ATCC 23769
Authors: Iyer Prashanti R. , Geib Scott M. , Catchmark Jeff , Kao Teh-hui , Tien Ming 
Published: June 2010

ABSTRACT

The Gram-negative bacterium
Gluconacetobacter hansenii
is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.
Transcriptional Activation of the Aldehyde Reductase YqhD by YqhC and Its Implication in Glyoxal Metabolism of
Escherichia coli
K-12
Authors: Lee Changhan , Kim Insook , Lee Junghoon , Lee Kang-Lok , Min Bumchan , Park Chankyu 
Published: June 2010

ABSTRACT

The reactive α-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated
in vivo
from sugars through oxidative stress. GO and MG are believed to be removed from cells by glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of GO-resistant (GO
r
) mutants from
Escherichia coli
strain MG1655 on LB plates containing 10 mM GO. By tagging the mutations with the transposon Tn
phoA
-132 and determining their cotransductional linkages, we were able to identify a locus to which most of the GO
r
mutations were mapped. DNA sequencing of the locus revealed that it contains the
yqhC
gene, which is predicted to encode an AraC-type transcriptional regulator of unknown function. The GO
r
mutations we identified result in missense changes in
yqhC
and were concentrated in the predicted regulatory domain of the protein, thereby constitutively activating the product of the adjacent gene
yqhD
. The transcriptional activation of
yqhD
by wild-type YqhC and its mutant forms was established by an assay with a β-galactosidase reporter fusion, as well as with real-time quantitative reverse transcription-PCR. We demonstrated that YqhC binds to the promoter region of
yqhD
and that this binding is abolished by a mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. YqhD facilitates the removal of GO through its NADPH-dependent enzymatic reduction activity by converting it to ethadiol via glycolaldehyde, as detected by nuclear magnetic resonance, as well as by spectroscopic measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as an aldehyde reductase with specificity for certain aldehydes, including GO.
Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed
Authors: Yosef Ido , Bochkareva Elena S. , Bibi Eitan 
Published: June 2010

ABSTRACT

The
Escherichia coli
signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since
E. coli
SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.

IMPORTANCE
The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited
in vitro
studies, it is believed that the
E. coli
SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several
in vivo
studies have suggested otherwise. To address this issue further
in vivo
, we utilized unbalanced conditions under which
E. coli
simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the
E. coli
SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.