bluuebunny/crossref_metadata_2025_split huggingface dataset:
VirB1 Orthologs fromBrucella suisand pKM101 Complement Defects of the Lytic Transglycosylase Required for Efficient Type IV Secretion fromAgrobacterium tumefaciens
Authors: Höppner Christoph , Liu Zhenying , Domke Natalie , Binns Andrew N. , Baron Christian 
Published: February 2004

ABSTRACTType IV secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells. The translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions. However, orthologs to proteins of the prototypical type IV system, VirB ofAgrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved. We here report a heterologous complementation in the case ofA. tumefaciens virB1defects with its orthologs fromBrucella suis(VirB1s) and the IncN plasmid pKM101 (TraL). In contrast, expression of the genes encoding the VirB1 orthologs from the IncF plasmid (open reading frame 169) and from theHelicobacter pylori cagpathogenicity island (HP0523) did not complement VirB1 functions. The complementation of VirB1 activity was assessed by T-pilus formation, by tumor formation on wounded plants, by IncQ plasmid transfer, and by IncQ plasmid recipient assay. Replacement of the key active-site Glu residue by Ala abolished the complementation by VirB1 fromB. suisand by TraL, demonstrating that heterologous complementation requires an intact lytic transglycosylase active site. In contrast, the VirB1 active-site mutant fromA. tumefaciensretained considerable residual activity in various activity assays, implying that this protein exerts additional effects during the type IV secretion process.
Role of Histone-Like Protein H-NS in Multidrug Resistance of
Escherichia coli
Authors: Nishino Kunihiko , Yamaguchi Akihito 
Published: February 2004

ABSTRACT

The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in
hns
result in various phenotypes, but the role of H-NS in the drug resistance of
Escherichia coli
has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the
hns
gene from the Δ
acrAB
mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the
hns
mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the
hns
deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene
tolC
. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of
acrEF
,
mdtEF
, and
emrKY
was observed in the Δ
hns
strain by quantitative real-time reverse transcription-PCR analysis. The Δ
hns
-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the
acrEF
gene greatly suppressed the level of Δ
hns
-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the
mdtEF
and
acrEF
genes completely suppressed Δ
hns
-mediated multidrug resistance, indicating that Δ
hns
-mediated multidrug resistance is due to derepression of the
acrEF
and
mdtEF
drug exporter genes.
TeiR, a LuxR-Type Transcription Factor Required for Testosterone Degradation inComamonas testosteroni
Authors: Pruneda-Paz José Luis , Linares Mauricio , Cabrera Julio E. , Genti-Raimondi Susana 
Published: February 2004

ABSTRACTWe have identified a new steroid-inducible gene (designatedteiR[testosterone-inducible regulator]) inComamonas testosteronithat is required for testosterone degradation. Nucleotide sequence analysis ofteiRpredicts a 391-amino-acid protein which shows homology between residues 327 and 380 (C-terminal domain) to the LuxR helix-turn-helix DNA binding domain and between residues 192 and 227 to the PAS sensor domain. This domain distribution resembles that described for TraR, a specific transcriptional regulator involved in quorum sensing inAgrobacterium tumefaciens. Analysis of the gene expression indicated thatteiRis tightly controlled at the transcriptional level by the presence of testosterone in the culture medium. AteiR-disrupted mutant strain was completely unable to use testosterone as the sole carbon and energy source. In addition, the expression of several steroid-inducible genes was abolished in this mutant. Northern blot assays revealed thatteiRis required for full expression ofsip48-β-HSD gene mRNA (encoding a steroid-inducible protein of 48 kDa and 3β-17β-hydroxysteroid dehydrogenase) and also of other steroid degradation genes, including those encoding 3α-hydroxysteroid dehydrogenase, Δ5-3-ketoisomerase, 3-oxo-steroid Δ1-dehydrogenase, and 3-oxo-steroid Δ4-(5α)-dehydrogenase enzymes. Moreover, whenteiRwas provided to theteiR-disrupted strain intrans, the transcription level of these genes was restored. These results indicate that TeiR positively regulates the transcription of genes involved in the initial enzymatic steps of steroid degradation inC. testosteroni.
Development of Surface Adhesion inCaulobacter crescentus
Authors: Bodenmiller Diane , Toh Evelyn , Brun Yves V. 
Published: February 2004

ABSTRACTCaulobacter crescentushas a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage,C. crescentusis often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment ofC. crescentusto abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion inC. crescentusis a complex developmental process. We found that the attachment ofC. crescentusto surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.
Transcriptional Profiling ofCaulobacter crescentusduring Growth on Complex and Minimal Media
Authors: Hottes Alison K. , Meewan Maliwan , Yang Desiree , Arana Naomi , Romero Pedro , McAdams Harley H. , Stephens Craig 
Published: February 2004

ABSTRACTMicroarray analysis was used to examine gene expression in the freshwater oligotrophic bacteriumCaulobacter crescentusduring growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential inC. crescentusfor growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth ofC. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.
Assembly of an Oxalate Decarboxylase Produced under σ
K
Control into the
Bacillus subtilis
Spore Coat
Authors: Costa Teresa , Steil Leif , Martins Lígia O. , Völker Uwe , Henriques Adriano O. 
Published: February 2004

ABSTRACT

Over 30 polypeptides are synthesized at various times during sporulation in
Bacillus subtilis
, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the
B. subtilis oxdD
gene was previously shown to have oxalate decarboxylase activity when it was produced in
Escherichia coli
and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of
oxdD
is induced during sporulation as a monocistronic unit under the control of σ
K
and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a
cotE
mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of
oxdD
resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.
Cardiolipin Domains in
Bacillus subtilis
Marburg Membranes
Authors: Kawai Fumitaka , Shoda Momoko , Harashima Rie , Sadaie Yoshito , Hara Hiroshi , Matsumoto Kouji 
Published: February 2004

ABSTRACT

Recently, use of the cardiolipin (CL)-specific fluorescent dye 10-
N
-nonyl-acridine orange (NAO) revealed CL-rich domains in the
Escherichia coli
membrane (E. Mileykovskaya and W. Dowhan, J. Bacteriol.
182:
1172-1175, 2000). Staining of
Bacillus subtilis
cells with NAO showed that there were green fluorescence domains in the septal regions and at the poles. These fluorescence domains were scarcely detectable in exponentially growing cells of the
clsA
-disrupted mutant lacking detectable CL. In sporulating cells with a wild-type lipid composition, fluorescence domains were observed in the polar septa and on the engulfment and forespore membranes. Both in the
clsA
-disrupted mutant and in a mutant with disruptions in all three of the paralogous genes (
clsA
,
ywjE
, and
ywiE
) for CL synthase, these domains did not vanish but appeared later, after sporulation initiation. A red shift in the fluorescence due to stacking of two dye molecules and the lipid composition suggested that a small amount of CL was present in sporulating cells of the mutants. Mass spectrometry analyses revealed the presence of CL in these mutant cells. At a later stage during sporulation of the mutants the frequency of heat-resistant cells that could form colonies after heat treatment was lower. The frequency of sporulation of these cells at 24 h after sporulation initiation was 30 to 50% of the frequency of the wild type. These results indicate that CL-rich domains are present in the polar septal membrane and in the engulfment and forespore membranes during the sporulation phase even in a
B. subtilis
mutant with disruptions in all three paralogous genes, as well as in the membranes of the medial septa and at the poles during the exponential growth phase of wild-type cells. The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranes are involved in sporulation.
Bordetella
Species Are Distinguished by Patterns of Substantial Gene Loss and Host Adaptation
Authors: Cummings C. A. , Brinig M. M. , Lepp P. W. , van de Pas S. , Relman D. A. 
Published: February 2004

ABSTRACT

Pathogens of the bacterial genus
Bordetella
cause respiratory disease in humans and animals. Although virulence and host specificity vary across the genus, the genetic determinants of this diversity remain unidentified. To identify genes that may underlie key phenotypic differences between these species and clarify their evolutionary relationships, we performed a comparative analysis of genome content in 42
Bordetella
strains by hybridization of genomic DNA to a microarray representing the genomes of three
Bordetella
species and by subtractive hybridization. Here we show that
B. pertussis
and
B. parapertussis
are predominantly differentiated from
B. bronchiseptica
by large, species-specific regions of difference, many of which encode or direct synthesis of surface structures, including lipopolysaccharide O antigen, which may be important determinants of host specificity. The species also exhibit sequence diversity at a number of surface protein-encoding loci, including the fimbrial major subunit gene,
fim2
. Gene loss, rather than gene acquisition, accompanied by the proliferation of transposons, has played a fundamental role in the evolution of the pathogenic bordetellae and may represent a conserved evolutionary mechanism among other groups of microbial pathogens.
Residues Required for
Bacillus subtilis
PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3
Authors: Chen Yinghua , Abdel-Fattah Wael R. , Hulett F. Marion 
Published: February 2004

ABSTRACT

Bacillus subtilis
PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP
I206A
, PhoP
R210A
, PhoP
L209A
, and PhoP
H208A
) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP
H205A
and PhoP
V204A
) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP
V202A
and PhoP
D203A
, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP
V190A
, PhoP
W191A
, PhoP
Y193A
, PhoP
F195A
, PhoP
G197A,
PhoP
T199A
, and PhoP
R200A
) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP
W191A
and PhoP
R200A
could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of
Escherichia coli
.
Genomic and Genetic Analysis of
Bordetella
Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes
Authors: Liu Minghsun , Gingery Mari , Doulatov Sergei R. , Liu Yichin , Hodes Asher , Baker Stephen , Davis Paul , Simmonds Mark , Churcher Carol , Mungall Karen , Quail Michael A. , Preston Andrew , Harvill Eric T. , Maskell Duncan J. , Eiserling Frederick A. , Parkhill Julian , Miller Jeff F. 
Published: February 2004

ABSTRACT

Liu et al. recently described a group of related temperate bacteriophages that infect
Bordetella
subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science
295:
2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the
mtd
(major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences,
bbp36
contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and
bpm
encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these
Bordetella
phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes.
Bordetella
bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection.
eBURST: Inferring Patterns of Evolutionary Descent among Clusters of Related Bacterial Genotypes from Multilocus Sequence Typing Data
Authors: Feil Edward J. , Li Bao C. , Aanensen David M. , Hanage William P. , Spratt Brian G. 
Published: February 2004

ABSTRACT

The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain
23F
-1 clonal complex of
Streptococcus pneumoniae
, “population snapshots” of the entire
S. pneumoniae
and
Staphylococcus aureus
MLST databases, and the more complicated clonal complexes observed for
Campylobacter jejuni
and
Neisseria meningitidis
.
Evolution of a Pathway to Novel Long-Chain Carotenoids
Authors: Umeno Daisuke , Arnold Frances H. 
Published: February 2004

ABSTRACT

Using methods of laboratory evolution to force the C
30
carotenoid synthase CrtM to function as a C
40
synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C
45
and C
50
carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in
Escherichia coli
comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.
Identification of a 349-Kilodalton Protein (Gli349) Responsible for Cytadherence and Glass Binding during Gliding of
Mycoplasma mobile
Authors: Uenoyama Atsuko , Kusumoto Akiko , Miyata Makoto 
Published: February 2004

ABSTRACT

Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of
Mycoplasma mobile
were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 ± 200. The antibody inhibited both the hemadsorption and glass binding of
M. mobile
. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.
The Switch I and II Regions of MinD Are Required for Binding and Activating MinC
Authors: Zhou Huaijin , Lutkenhaus Joe 
Published: February 2004

ABSTRACTMinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations inminDand carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.