bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutagenesis of
Neisseria meningitidis
by In Vitro Transposition of
Himar1 mariner
Authors: Pelicic Vladimir , Morelle Sandrine , Lampe David , Nassif Xavier 
Published: July 2002

ABSTRACT

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of
Neisseria meningitidis
. Here, we demonstrate that in vitro
Himar1 mariner
transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into
N. meningitidis
by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the
Himar1
insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that
Himar1
transposed randomly in
N. meningitidis
. Taken together, these data demonstrate that
Himar1
in vitro transposition can lead to the exhaustive mutagenesis of
N. meningitidis
, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.
Characterization and Expression of HmuR, a TonB-Dependent Hemoglobin Receptor of
Porphyromonas gingivalis
Authors: Simpson Waltena , Olczak Teresa , Genco Caroline Attardo 
Published: July 2002

ABSTRACT

The gram-negative pathogen
Porphyromonas gingivalis
requires hemin for growth. Hemoglobin bound to haptoglobin and hemin complexed to hemopexin can be used as heme sources, indicating that
P. gingivalis
must have a means to remove the hemin from these host iron-binding proteins. However, the specific mechanisms utilized by
P. gingivalis
for the extraction of heme from heme-binding proteins and for iron transport are poorly understood. In this study we have determined that a newly identified TonB-dependent hemoglobin-hemin receptor (HmuR) is involved in hemoglobin binding and utilization in
P. gingivalis
A7436. HmuR shares amino acid homology with TonB-dependent outer membrane receptors of gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin, including HemR of
Yersinia enterocolitica
, ShuA of
Shigella dysenteriae
, HpuB of
Neisseria gonorrhoeae
and
N. meningitidis
, HmbR of
N. meningitidis
, HgbA of
Haemophilus ducreyi
, and HgpB of
H. influenzae
. Southern blot analysis confirmed the presence of the
hmuR
gene and revealed genetic variability in the carboxy terminus of
hmuR
in
P. gingivalis
strains 33277, 381, W50, and 53977. We also identified directly upstream of the
hmuR
gene a gene which we designated
hmuY
. Upstream of the
hmuY
start codon, a region with homology to the Fur binding consensus sequence was identified. Reverse transcription-PCR analysis revealed that
hmuR
and
hmuY
were cotranscribed and that transcription was negatively regulated by iron. Inactivation of
hmuR
resulted in a decreased ability of
P. gingivalis
to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sources.
Escherichia coli
cells expressing recombinant HmuR were shown to bind hemoglobin and hemin. Furthermore, purified recombinant HmuR was demonstrated to bind hemoglobin. Taken together, these results indicate that HmuR serves as the major TonB-dependent outer membrane receptor involved in the utilization of both hemin and hemoglobin in
P. gingivalis
.
Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for
p
-Hydroxybenzoate Polyprenyl Diphosphate Transferase
Authors: Uchida Naonori , Suzuki Kengo , Saiki Ryoichi , Kainou Tomohiro , Tanaka Katsunori , Matsuda Hideyuki , Kawamukai Makoto 
Published: July 2002

ABSTRACT

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps.
p
-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated
ppt1
) encoding PHB polyprenyl diphosphate transferase from
Schizosaccharomyces pombe
and constructed a strain with a disrupted
ppt1
gene. This strain could not grow on minimal medium supplemented with glucose. Expression of
COQ2
from
Saccharomyces cerevisiae
in the defective
S. pombe
strain restored growth and enabled the cells to produce ubiquinone-10, indicating that
COQ2
and
ppt1
are functional homologs. The
ppt1
-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and α-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the
ppt1
-deficient strain is sensitive to H
2
O
2
and Cu
2+
. Interestingly, we also found that the
ppt1
-deficient strain produced a significant amount of H
2
S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in
S. pombe
. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in
S. pombe
. Thus, analysis of the phenotypes of
S. pombe
strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.
Intergeneric Communication in Dental Plaque Biofilms
Authors: Xie Hua , Cook Guy S. , Costerton J. William , Bruce Greg , Rose Timothy M. , Lamont Richard J. 
Published: July 2002

ABSTRACT

Dental plaque is a complex biofilm that accretes in a series of discrete steps proceeding from a gram-positive streptococcus-rich biofilm to a structure rich in gram-negative anaerobes. This study investigated information flow between two unrelated plaque bacteria,
Streptococcus cristatus
and
Porphyromonas gingivalis
. A surface protein of
S. cristatus
caused repression of the
P. gingivalis
fimbrial gene (
fimA
), as determined by a chromosomal
fimA
promoter-
lacZ
reporter construct and by reverse transcription-PCR. Signaling activity was associated with a 59-kDa surface protein of
S. cristatus
and showed specificity for the
fimA
gene. Furthermore,
P. gingivalis
was unable to form biofilm microcolonies with
S. cristatus
. Thus,
S. cristatus
is capable of modulating virulence gene expression in
P. gingivalis
, consequently influencing the development of pathogenic plaque.
Quorum Sensing but Not Autoinduction of Ti Plasmid Conjugal Transfer Requires Control by the Opine Regulon and the Antiactivator TraM
Authors: Piper Kevin R. , Farrand Stephen K. 
Published: July 2002

ABSTRACT

Conjugal transfer of the Ti plasmids from
Agrobacterium tumefaciens
is controlled by autoinduction via the transcriptional activator TraR and the acyl-homoserine lactone ligand,
Agrobacterium
autoinducer (AAI). This control process is itself regulated by opines, which are small carbon compounds produced by the crown gall tumors that are induced by the bacteria. Opines control autoinduction by regulating the expression of
traR
. Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population level of 10
7
cells per cm
2
. Donors incubated with the opines and AAI transferred their Ti plasmids at population levels about 10-fold lower than those incubated with opines only. Transcription of the
tra
regulon, as assessed by monitoring a
traA
::
lacZ
reporter, showed a similar dependence on the density of the donor population. However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag of between 15 and 20 h in the development of conjugal competence. Moreover, even after this latent period, maximal transfer frequencies required several hours to develop. This lag period was independent of the population density of the donors but could be reduced somewhat by addition of exogenous AAI. Quorum-dependent development of conjugal competence required control by the opine regulon; donors harboring a mutant of pTiC58 deleted for the master opine responsive repressor
accR
transferred the Ti plasmid at maximum frequencies at very low population densities. Similarly, an otherwise wild-type derivative of pTiC58 lacking
traM
, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-independent manner in the absence of the conjugal opines. Thus, while quorum sensing is dependent upon autoinduction, the two phenomena are not synonymous. We conclude that conjugal transfer of pTiC58 is regulated in a quorum-dependent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accumulation of AAI but also the expression of TraR and the synthesis of this protein at levels that overcome the inhibitory activity of TraM.
Roles for the
Rhodobacter sphaeroides
CcmA and CcmG Proteins
Authors: Cox Rebecca L. , Patterson Chandra , Donohue Timothy J. 
Published: July 2002

ABSTRACT

Rhodobacter sphaeroides
cells containing an in-frame deletion within
ccmA
lack detectable soluble and membrane-bound
c
-type cytochromes and are unable to grow under conditions where these proteins are required. Only strains merodiploid for
ccmABCDG
were found after attempting to generate cells containing either a
ccmG
null mutation or a
ccmA
allele that should be polar on to expression of
ccmBCDG
, suggesting that CcmG has another important role in
R. sphaeroides
.
Fructose Uptake in
Sinorhizobium meliloti
Is Mediated by a High-Affinity ATP-Binding Cassette Transport System
Authors: Lambert Annie , Østerås Magne , Mandon Karine , Poggi Marie-Christine , Le Rudulier Daniel 
Published: July 2002

ABSTRACT

By transposon mutagenesis, we have isolated a mutant of
Sinorhizobium meliloti
which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the Tn
phoA
insertion revealed the presence of six open reading frames (ORFs) organized in two loci,
frcRS
and
frcBCAK
, transcribed divergently. The
frcBCA
genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (

K
m

of 6 μM) fructose uptake system in
S. meliloti
. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of
S. meliloti frcBCAK
in
Escherichia coli
, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in
S. meliloti
. The expression of the
frcBC
genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the Tn
phoA
mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is expressed in bacteroids isolated from alfalfa nodulated by
S. meliloti
wild-type strains.
The Phosphate-Binding Protein of
Escherichia coli
Is Not Essential for P
i
-Regulated Expression of the
pho
Regulon
Authors: Hoffer Sally M. , Tommassen Jan 
Published: July 2002

ABSTRACT

Disruption of
pstS
encoding the P
i
-binding protein in
Escherichia coli
generally leads to the constitutive expression of the
pho
regulon. We demonstrate that P
i
-controlled expression is restored when the activity of the P
i
transporter PitA or PitB is increased. Apparently, PstS is not an essential component of the signal transduction pathway.
Regulation of
d
-Alanyl-Lipoteichoic Acid Biosynthesis in
Streptococcus agalactiae
Involves a Novel Two-Component Regulatory System
Authors: Poyart Claire , Lamy Marie Cécile , Boumaila Claire , Fiedler Franz , Trieu-Cuot Patrick 
Published: July 2002

ABSTRACT

The
dlt
operon of gram-positive bacteria comprises four genes (
dltA, dltB, dltC
, and
dltD
) that catalyze the incorporation of
d
-alanine residues into the lipoteichoic acids (LTAs). In this work, we characterized the
dlt
operon of
Streptococcus agalactiae
, which, in addition to the
dltA
to
dltD
genes, included two regulatory genes, designated
dltR
and
dltS
, located upstream of
dltA
. The
dltR
gene encodes a 224-amino-acid putative response regulator belonging to the OmpR family of regulatory proteins. The
dltS
gene codes for a 395-amino-acid putative histidine kinase thought to be involved in the sensing of environmental signals. The
dlt
operon of
S. agalactiae
is mainly transcribed from the P

dltR

promoter, which directs synthesis of a 6.5-kb transcript encompassing
dltR, dltS, dltA, dltB, dltC
, and
dltD
, and from a weaker promoter, P

dltA

, which is located in the 3′ extremity of
dltS
. We demonstrate that P

dltR

, but not P

dlA

, is activated by DltR in the presence of DltS in
d
-Ala-deficient LTA mutants resulting from insertional inactivation of the
dltA
gene, which encodes the cytoplasmic
d
-alanine-
d
-alanyl carrier ligase DltA. Expression of the
dlt
operon does not require DltR and DltS, since the basal activity of P

dltR

is high, being 20-fold that of the constitutive promoter P

aphA-3

which directs synthesis of the kanamycin resistance gene
aphA-3
in various gram-positive bacteria. We hypothesize that the role of DltR and DltS in the control of expression of the
dlt
operon is to maintain the level of
d
-Ala esters in LTAs at a constant and appropriate value whatever the environmental conditions. The DltA
−
mutant displayed the ability to form clumps in standing culture and exhibited an increased susceptibility to the cationic antimicrobial polypeptide colistin.
Vibrio fischeriOuter Membrane Protein OmpU Plays a Role in Normal Symbiotic Colonization
Authors: Aeckersberg F. , Lupp C. , Feliciano B. , Ruby E. G. 
Published: July 2002

ABSTRACTThe nascent light-emitting organ of newly hatched juveniles of the Hawaiian sepiolid squidEuprymna scolopesis specifically colonized by cells ofVibrio fischerithat are obtained from the ambient seawater. The mechanisms that promote this specific, cooperative colonization are likely to require a number of bacterial and host-derived factors and activities, only some of which have been described to date. A characteristic of many host-pathogen associations is the presence of bacterial mechanisms that allow attachment to specific tissues. These mechanisms have been well characterized and often involve bacterial fimbriae or outer membrane proteins (OMPs) that act as adhesins, the expression of which has been linked to virulence regulators such as ToxR inVibrio cholerae. Analogous or even homologous mechanisms are probably operative in the initiation and persistence of cooperative bacterial associations, although considerably less is known about them. We report the presence inV. fischeriofompU, a gene encoding a 32.5-kDa protein homolog of two other OMPs, OmpU ofV. cholerae(50.8% amino acid sequence identity) and OmpL ofPhotobacterium profundum(45.5% identity). A null mutation introduced into theV. fischeri ompUresulted in the loss of an OMP with an estimated molecular mass of about 34 kDa; genetic complementation of the mutant strain with a DNA fragment containing only theompUgene restored the production of this protein. The expression of theV. fischeriOmpU was not significantly affected by either (i) iron or phosphate limitation or (ii) a mutation that rendersV. fischeridefective in the synthesis of a homolog of the OMP-regulatory protein ToxR. TheompUmutant grew normally in complex nutrient media but was more susceptible to growth inhibition in the presence of either anionic detergents or the antimicrobial peptide protamine sulfate. Interestingly, colonization experiments showed that theompUnull mutant initiated a symbiotic association with juvenile light organ tissue with only about 60% of the effectiveness of the parent strain. When colonization did occur, it proceeded more slowly and resulted in an approximately fourfold-smaller bacterial population. Surprisingly, there was no evidence that in a mixed infection with its parent, theompU-defective strain had a competitive disadvantage, suggesting that the presence of the parent strain provided a shared compensatory activity. Thus, the OmpU protein appears to play a role in the normal process by whichV. fischeriinitiates its colonization of the nascent light organ of juvenile squids.
Differential Roles of the
Pseudomonas aeruginosa
PA14
rpoN
Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice
Authors: Hendrickson Erik L. , Plotnikova Joulia , Mahajan-Miklos Shalina , Rahme Laurence G. , Ausubel Frederick M. 
Published: July 2002

ABSTRACT

We cloned the
rpoN (ntrA
,
glnF)
gene encoding the alternate sigma factor ς
54
from the opportunistic multihost pathogen
Pseudomonas aeruginosa
strain PA14. A marker exchange protocol was used to construct the PA14
rpoN
insertional mutation
rpoN
::Gen
r
. PA14
rpoN
::Gen
r
synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of
rpoN
mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14,
rpoN
::Gen
r
was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode
Caenorhabditis elegans
. In an
Arabidopsis thaliana
leaf infectivity assay, although
rpoN
::Gen
r
exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in
Arabidopsis
leaves, it nevertheless elicited similar disease symptoms to wild-type
P. aeruginosa
PA14 at later stages of infection.
rpoN
::Gen
r
was not impaired in virulence in a
Galleria mellonella
(greater wax moth) pathogenicity model. These data indicate that
rpoN
does not regulate the expression of any genes that encode virulence factors universally required for
P. aeruginosa
pathogenicity in diverse hosts.
Degenerative Minimalism in the Genome of a Psyllid Endosymbiont
Authors: Clark Marta A. , Baumann Linda , Thao MyLo Ly , Moran Nancy A. , Baumann Paul 
Published: July 2002

ABSTRACT

Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of
Carsonella ruddii
, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont
Buchnera aphidicola
. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of
C. ruddii
, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of
C. ruddii
are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the
C. ruddii
genome is not subject to constraints that limit the evolution of other known eubacteria.
MeaA, a Putative Coenzyme B
12
-Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in
Streptomyces cinnamonensis
Authors: Zhang Weiwen , Reynolds Kevin A. 
Published: July 2002

ABSTRACT

The ratio of the major monensin analogs produced by
Streptomyces cinnamonensis
is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The
meaA
gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B
12
-dependent mutases. Plasmid-based expression of
meaA
from the
ermE
∗ promoter in the
S. cinnamonensis
C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a
meaA
mutant,
S. cinnamonensis
WM2 (generated from the C730.1 strain by insertional inactivation of
meaA
by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an
S. cinnamonensis
WD2 strain (an
icm meaA
mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the
meaA
gene or the
Amycolatopsis mediterranei mutAB
genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the
meaA
gene product is significantly involved in methylmalonyl-CoA production in
S. cinnamonensis
and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.