bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Identification of Oligoalginate Lyase of
Sphingomonas
sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate
Authors: Hashimoto Wataru , Miyake Osamu , Momma Keiko , Kawai Shigeyuki , Murata Kousaku
Published: July 2002

A bacterium,
Sphingomonas
sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in
Sphingomonas
sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (
aly
) and the ABC transporter (
algS
,
algM1
, and
algM2
). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.
A Homologue of an Operon Required for DNA Transfer in
Agrobacterium
Is Required in
Brucella abortus
for Virulence and Intracellular Multiplication
Authors: Sieira Rodrigo , Comerci Diego J. , Sánchez Daniel O. , Ugalde Rodolfo A.
Published: July 2002

As part of a
Brucella abortus
2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (
virB
) highly homologous to bacterial type IV secretion systems. The
B. abortus virB
locus is a collinear arrangement of 13 open reading frames (ORFs). Between
virB1
and
virB2
and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of
Brucella
intergenic regions were found. Gene reporter studies demonstrated that the
B. abortus virB
locus constitutes an operon transcribed from
virB1
which is turned on during the stationary phase of growth. A
B. abortus
polar
virB1
mutant failed to replicate in HeLa cells, indicating that the
virB
operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in
virB10
showed different behaviors in mice and in the HeLa cell infection assay, suggesting that
virB10
per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the
virB
operon constitutes a major determinant of
B. abortus
virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of
B. abortus
to an endoplasmic reticulum-related replication compartment.
The
Pseudomonas aeruginosa
Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS
Authors: Winzer Klaus , Falconer Colin , Garber Nachman C. , Diggle Stephen P. , Camara Miguel , Williams Paul
Published: July 2002

In
Pseudomonas aeruginosa
, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators,
N
-(3-oxododecanoyl)-
l
-homoserine lactone (3O-C12-HSL) and
N
-butanoyl-
l
-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the
rhl
locus while, in a
lasR
mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene,
lecA
, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the
lecA
translational start codon. A
lux
box-type element together with RpoS (ς
S
) consensus sequences was identified upstream of the putative promoter region. In
Escherichia coli
, expression of a
lecA
::
lux
reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in
P. aeruginosa
PAO1, the expression of a chromosomal
lecA
::
lux
fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of
rpoS
abolished lectin synthesis in
P. aeruginosa
, demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the
lecA
::
lux
reporter in
E. coli
could be inhibited by the presence of 3O-C12-HSL, this did not occur in
P. aeruginosa
. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of
lecA
expression.
Structural Characterization of the Released Polysaccharide of Desiccation-Tolerant
Nostoc commune
DRH-1
Authors: Helm Richard F. , Huang Zebo , Edwards Devin , Leeson Heidi , Peery William , Potts Malcolm
Published: July 2002

The structure of the viscous extracellular polysaccharide (glycan) of desiccation-tolerant
Nostoc commune
DRH-1 was determined through chromatographic and spectroscopic methods. The polysaccharide is novel in that it possesses a 1-4-linked xylogalactoglucan backbone with
d
-ribofuranose and 3-
O
-[(
R
)-1-carboxyethyl]-
d
-glucuronic acid (nosturonic acid) pendant groups. The presence of
d
-ribose and nosturonic acid as peripheral groups is unusual, and their potential roles in modulating the rheological properties of the glycan are discussed. Nosturonic acid was present in the glycans of
N. commune
from diverse geographic locations, suggesting that this uronic acid is an integral component of this cosmopolitan anhydrophile.
Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme
Authors: Parales Rebecca E. , Lee Kyoung , Resnick Sol M. , Jiang Haiyan , Lessner Daniel J. , Gibson David T.
Published: July 2002

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by
Pseudomonas
sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes
cis
-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of
cis
-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1
R
,2
S
], compared to the enantiomerically pure [>99% (+)-1
R
,2
S
] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.
The
Staphylococcus aureus lrgAB
Operon Modulates Murein Hydrolase Activity and Penicillin Tolerance
Authors: Groicher Kajetan H. , Firek Brian A. , Fujimoto David F. , Bayles Kenneth W.
Published: July 2002

Previous studies in our laboratory have shown that the
Staphylococcus aureus
LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a
lrgAB
mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the
lrgAB
mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the
lrgAB
defect by providing the
lrgAB
genes in
trans
restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the
lrgAB
mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the
lrgAB
mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the
lrgAB
operon was shown to be maximally expressed. This effect of the
lrgAB
mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the
lrgAB
mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which
lrgAB
expression was shown to be minimal. However, expression of the
lrgAB
operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of
S. aureus
involves a novel regulator of murein hydrolase activity.
Characterization of the
Mycobacterium tuberculosis iniBAC
Promoter, a Promoter That Responds to Cell Wall Biosynthesis Inhibition
Authors: Alland David , Steyn Andries J. , Weisbrod Torin , Aldrich Kate , Jacobs William R.
Published: July 2002

The cell wall provides an attractive target for antibiotics against
Mycobacterium tuberculosis
. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (
iniB
,
iniA
, and
iniC
) organized in tandem on the
M. tuberculosis
genome. Here, we investigate the unique features of the putative
iniBAC
promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the
iniBAC
promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the
iniBAC
promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in
M. tuberculosis
and other mycobacteria.
Characterization of In53, a Class 1 Plasmid- and Composite Transposon-Located Integron ofEscherichia coliWhich Carries an Unusual Array of Gene Cassettes
Authors: Naas Thierry , Mikami Yuzuru , Imai Tamae , Poirel Laurent , Nordmann Patrice
Published: July 2002

Further characterization of the genetic environment of the gene encoding theEscherichia coliextended-spectrum β-lactamase,blaVEB-1, revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition toblaVEB-1. While theaadBand thearr-2gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of thecmlAfamily, (ii) aqacallele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette,aacA1b/orfG,which encodes a novel 6′-N-acetyltransferase, and (iv) a fused gene cassette,oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition,oxa10andaadA1genes were expressed from their own promoter sequence present upstream of theoxa10cassette.arr-2coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by thearr-1gene fromMycobacterium smegmatisDSM43756. While inM. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered fromE. coliculture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26insertion sequence, which was also present in the 3′ conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.
Characterization of
fsr
, a Regulator Controlling Expression of Gelatinase and Serine Protease in
Enterococcus faecalis
OG1RF
Authors: Qin Xiang , Singh Kavindra V. , Weinstock George M. , Murray Barbara E.
Published: July 2002

We have previously identified a locus,
fsr
, a homologue of staphylococcal
agr
loci, which positively regulates the expression of gelatinase and serine protease (encoded by
gelE
and
sprE
, respectively) in
Enterococcus faecalis
OG1RF. The expression of the three genes in the
fsr
locus,
fsrA
,
fsrB
, and
fsrC
, appears to be autoregulated, and we have shown that mutants with insertion disruptions in each of these three genes were significantly attenuated in a mouse peritonitis model compared to the parent strain. In the present study, we showed that
fsrB
and
fsrC
are highly expressed in the postexponential growth phase and that their expression is cell density dependent. Reverse transcriptase PCR using primers covering the intergenic regions in the
fsr
/
gelE
loci confirmed that
fsrB
and
fsrC
, as well as
gelE
and
sprE
, are cotranscribed. We also showed, using a nonpolar
fsrB
deletion mutant, that
fsrB
, the homologue of
agrB
of staphylococci with unknown function, is required for the regulatory function of
fsr
. Primer extension and analysis of transcriptional fusions indicated the presence of promoters immediately upstream of
fsrA
, of
fsrB
, and of
gelE
and that the
fsrB
and
gelE
promoters are
fsr
dependent, while the
fsrA
promoter is an
fsr
-independent weak constitutive promoter. Two conserved 7-bp direct repeats were found immediately upstream of the
fsrB
and
gelE
promoters, similar to the repeats found upstream of P2 and P3 promoters of the
agr
locus; deletions and mutations in the repeated sequences completely abolished the
fsrB
and
gelE
promoter activities, suggesting that the repeats are important for the regulatory function in the
fsrB
and
gelE
promoter regions.
Identification of a Dedicated Recycling Pathway for Anhydro-
N
-Acetylmuramic Acid and
N
-Acetylglucosamine Derived from
Escherichia coli
Cell Wall Murein
Authors: Park James T.
Published: July 2002

Turnover and recycling of the cell wall murein represent a major metabolic pathway of
Escherichia coli
. It is known that
E. coli
efficiently reuses, i.e., recycles, its murein tripeptide,
l
-alanyl-γ-
d
-glutamyl-
meso
-diaminopimelate, to form new murein. However, the question of whether the cells also recycle the amino sugar moieties of cell wall murein has remained unanswered. It is demonstrated here that
E. coli
recycles the
N
-acetylglucosamine present in cell wall murein degradation products for de novo murein and lipopolysaccharide synthesis. Furthermore,
E. coli
also recycles the anhydro-
N
-acetylmuramic acid moiety by first converting it into
N
-acetylglucosamine. Based on the results obtained by studying mutants unable to recycle amino sugars, the pathway for recycling is revealed.
Alginate Overproduction AffectsPseudomonas aeruginosaBiofilm Structure and Function
Authors: Hentzer Morten , Teitzel Gail M. , Balzer Grant J. , Heydorn Arne , Molin Søren , Givskov Michael , Parsek Matthew R.
Published: July 2002

During the course of chronic cystic fibrosis (CF) infections,Pseudomonas aeruginosaundergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. ChronicP. aeruginosainfections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginate-overproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.
Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from
Neisseria gonorrhoeae
Authors: Pagotto Franco , Dillon Jo-Anne R.
Published: July 2002

The β-lactamase-producing Asia-type plasmid pJD4 of
Neisseria gonorrhoeae
is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both
N. gonorrhoeae
and
Haemophilus ducreyi
. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named
ori1, ori2
, and
ori3
. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when
ori2
and
ori3
are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only
ori1
, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack
ori1
but carry
ori2
and
ori3
do not require Pol I and are incompatible with IncW plasmids, suggesting that the
ori2
or
ori3
region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for
ori2
and
ori3
to function. This Rep protein is distinct from RepA, which is necessary for
ori1.
Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins.
Type II Secretion by
Aeromonas salmonicida
: Evidence for Two Periplasmic Pools of Proaerolysin
Authors: Burr Sarah E. , Diep Dzung B. , Buckley J. Thomas
Published: July 2002

Aeromonas salmonicida
containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of
aerA
led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by
A. salmonicida
.
Functional Analysis of Genes for Biosynthesis of Pyocyanin and Phenazine-1-Carboxamide from
Pseudomonas aeruginosa
PAO1
Authors: Mavrodi Dmitri V. , Bonsall Robert F. , Delaney Shannon M. , Soule Marilyn J. , Phillips Greg , Thomashow Linda S.
Published: July 2002

Two seven-gene phenazine biosynthetic loci were cloned from
Pseudomonas aeruginosa
PAO1. The operons, designated
phzA1B1C1D1E1F1G1
and
phzA2B2C2D2E2F2G2
, are homologous to previously studied phenazine biosynthetic operons from
Pseudomonas fluorescens
and
Pseudomonas aureofaciens
. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from
P. aeruginosa
is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in
P. aeruginosa
by two novel phenazine-modifying genes,
phzM
and
phzS
, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of
phzS
alone in
Escherichia coli
or in enzymes, pyocyanin-nonproducing
P. fluorescens
resulted in conversion of PCA to 1-hydroxyphenazine.
P. aeruginosa
with insertionally inactivated
phzM
or
phzS
developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene,
phzH
, which has a homologue in
Pseudomonas chlororaphis
, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in
P. aeruginosa
PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in
P. aeruginosa
consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.
MexR Repressor of the
mexAB-oprM
Multidrug Efflux Operon of
Pseudomonas aeruginosa
: Identification of MexR Binding Sites in the
mexA-mexR
Intergenic Region
Authors: Evans Kelly , Adewoye Lateef , Poole Keith
Published: July 2002

The MexR repressor of the
mexAB-oprM
multidrug efflux operon of
Pseudomonas aeruginosa
was purified as a C-terminal histidine-tagged protein by metal chelate affinity chromatography. The purified protein was shown to bind ca. 200 bp upstream of
mexA
, at two sites, each of which contains a repeat of the nucleotide sequence GTTGA in inverse orientation. DNA sequence analysis identified
mexA
and
mexR
promoters within the MexR binding regions, consistent with the previously observed negative regulation of
mexR
and
mexAB-oprM
expression by MexR. Transcription of
mexA
from the promoter originating within the MexR binding site II was confirmed and shown to be markedly enhanced in a
nalB
(i.e.,
mexR
) mutant of
P. aeruginosa
. A second
mexA
promoter was also identified, ca. 70 bp upstream of
mexAB-oprM
, and transcription from this promoter appeared to occur in both the wild type and a
nalB
mutant. Production of MexAB-OprM in wild-type cells may be due to expression from a constitutively expressed proximal promoter, while MexAB-OprM hyperexpression in
nalB
mutants is due to the additional expression from a MexR-regulated distal promoter.
Bap, a
Staphylococcus aureus
Surface Protein Involved in Biofilm Formation
Authors: Cucarella Carme , Solano Cristina , Valle Jaione , Amorena Beatriz , Lasa Íñigo , Penadés José R.
Published: July 2002

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing
Staphylococcus aureus
isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (
bap
[for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (
Pseudomonas aeruginosa
and
Salmonella enterica
serovar Typhi) and gram-positive (
Enteroccocus faecalis
) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids.
bap
was present in a small fraction of bovine mastitis isolates (5% of the 350
S. aureus
isolates tested), but it was absent from the 75 clinical human
S. aureus
isolates analyzed. All staphylococcal isolates harboring
bap
were highly adherent and strong biofilm producers. In a mouse infection model
bap
was involved in pathogenesis, causing a persistent infection.
Overexpression of Two Different GTPases Rescues a Null Mutation in a Heat-Induced rRNA Methyltransferase
Authors: Tan Jacqueline , Jakob Ursula , Bardwell James C. A.
Published: July 2002

The
Escherichia coli
RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the
rrmJ
(
ftsJ
) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other
E. coli
genes can restore the severe growth defect observed in
rrmJ
null mutants, we constructed an overexpression library from the
rrmJ
deletion strain and cloned and identified the
E. coli
genes that were capable of rescuing the
rrmJ
mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the
rrmJ
deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an
rrmJ
strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.
Behavior of Sister Copies of Mini-F Plasmid after Synchronized Plasmid Replication inEscherichia coliCells
Authors: Onogi Toshinari , Miki Takeyoshi , Hiraga Sota
Published: July 2002

To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sisteroriCcopies of theEscherichia colichromosome.
Northern, Morphological, and Fermentation Analysis of
spo0A
Inactivation and Overexpression in
Clostridium acetobutylicum
ATCC 824
Authors: Harris Latonia M. , Welker Neil E. , Papoutsakis Eleftherios T.
Published: July 2002

The
Clostridium acetobutylicum
ATCC 824
spo0A
gene was cloned, and two recombinant strains were generated, an
spo0A
inactivation strain (SKO1) and an
spo0A
overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration—a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No
spo0A
transcripts were detected in SKO1, while transcription of two solvent formation operons (
aad-ctfA-ctfB
and
adc
; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated
spo0A
transcript levels and displayed a bimodal pattern of
spo0A
transcription similar to that of
B. subtilis.
Microscopic studies indicated that sporulation was both enhanced and accelerated due to
spo0A
overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (
aad-ctfA-ctfB
and
adc
) and three sporulation-specific genes (
spoIIGA
,
sigE
, and
sigG
) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.
Toxic Waste Disposal in
Escherichia coli
Authors: Helling Robert B. , Janes Brian K. , Kimball Heather , Tran Timothy , Bundesmann Michael , Check Pietra , Phelan Darcy , Miller Charles
Published: July 2002

About 10% of the nalidixic acid-resistant (Nal
r
) mutants in a transposition-induced library exhibited a growth factor requirement as the result of
cysH
,
icdA
,
metE
, or
purB
mutation. Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role. Transcription of
acrAB
was increased in each type of Nal
r
mutant. In the
icdA
and
purB
mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump. The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism.
Microviridae
, a Family Divided: Isolation, Characterization, and Genome Sequence of φMH2K, a Bacteriophage of the Obligate Intracellular Parasitic Bacterium
Bdellovibrio bacteriovorus
Authors: Brentlinger Karie L. , Hafenstein Susan , Novak Christopher R. , Fane Bentley A. , Borgon Robert , McKenna Robert , Agbandje-McKenna Mavis
Published: July 2002

A novel single-stranded DNA phage, φMH2K, of
Bdellovibrio bacteriovorus
was isolated, characterized, and sequenced. This phage is a member of the
Microviridae
, a family typified by bacteriophage φX174. Although
B. bacteriovorus
and
Escherichia coli
are both classified as proteobacteria, φMH2K is only distantly related to φX174. Instead, φMH2K exhibits an extremely close relationship to the
Microviridae
of
Chlamydia
in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages.
Pseudomonas aeruginosa
Displays Multiple Phenotypes during Development as a Biofilm
Authors: Sauer Karin , Camper Anne K. , Ehrlich Garth D. , Costerton J. William , Davies David G.
Published: July 2002

Complementary approaches were employed to characterize transitional episodes in
Pseudomonas aeruginosa
biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic
P. aeruginosa
were compared with planktonic cultures of
Pseudomonas putida
. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that
P. aeruginosa
displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.
Salicylate 5-Hydroxylase from
Ralstonia
sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase
Authors: Zhou Ning-Yi , Al-Dulayymi Jumáa , Baird Mark S. , Williams Peter A.
Published: July 2002

The genes from the oxygenase cluster
nagAaGHAbAcAd
of naphthalene-degrading
Ralstonia
sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed
nagAaGHAb
or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the
nag
naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the
nag
proteins and the corresponding
nah
NDO proteins from the “classical” naphthalene degrader
P. putida
NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.
nblS, a Gene Involved in Controlling Photosynthesis-Related Gene Expression during High Light and Nutrient Stress inSynechococcus elongatusPCC 7942
Authors: van Waasbergen Lorraine G. , Dolganov Nadia , Grossman Arthur R.
Published: July 2002

The HliA protein of the cyanobacteriumSynechococcus elongatusPCC 7942 is a small, thylakoid-associated protein that appears to play a role in photoprotection; its transcript rapidly accumulates in response to high-intensity light (HL) and thehligene family is required for survival of cells in high light. In order to discover regulatory factors involved in HL acclimation in cyanobacteria, a screen was performed for chemically generated mutants unable to properly control expression of thehliAgene in response to HL. One such mutant was identified, and complementation analysis led to the identification of the affected gene, designatednblS. Based on its deduced protein sequence, NblS appears to be a membrane-bound, PAS-domain-bearing, sensor histidine kinase of two-component regulatory systems in bacteria. ThenblSmutant was unable to properly control light intensity-mediated expression of several other photosynthesis-related genes, including all threepsbAgenes and thecpcBAgenes. The mutant was also unable to control expression of thehliAandpsbAgenes in response to low-intensity blue/UV-A light, a response that may be related to the HL-mediated regulation of the genes. Additionally, in response to nutrient deprivation, thenblSmutant was unable to properly control accumulation of thenblAtranscript and associated degradation of the light-harvesting phycobilisomes. ThenblSmutant dies more rapidly than wild-type cells following exposure to HL or nutrient deprivation, likely due to its inability to properly acclimate to these stress conditions. Thus, the NblS protein is involved in the control of a number of processes critical for altering the photosynthetic apparatus in response to both HL and nutrient stress conditions.
Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the
crmB
Gene Region
Authors: Loparev Vladimir N. , Massung Robert F. , Esposito Joseph J. , Meyer Hermann
Published: July 2002

A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (
crmB
) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated
crmB
gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.
Performance of Applied Biosystems ViroSeq HIV-1 Genotyping System for Sequence-Based Analysis of Non-Subtype B Human Immunodeficiency Virus Type 1 from Uganda
Authors: Mracna Martin , Becker-Pergola Graziella , Dileanis Joann , Guay Laura A. , Cunningham Shawn , Jackson J. Brooks , Eshleman Susan H.
Published: July 2002

The Applied Biosystems ViroSeq HIV-1 Genotyping System is a commercially available, integrated system for sequence-based analysis of drug resistance mutations in human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for analysis of non-subtype B HIV-1 by analyzing plasma samples from Ugandan women and infants. Plasma samples were obtained from 105 women and 25 infants enrolled in a Ugandan clinical trial. HIV-1 analysis was performed with the ViroSeq system according to the manufacturer's instructions, except that the volume of plasma used for analysis was less than the recommended 0.5 ml for some samples. Viral loads ranged from 2,313 to 2,336,400 copies/ml. PCR products suitable for sequencing were amplified from all samples tested. Complete sequences for protease (amino acids 1 to 99) and RT (amino acids 1 to 320) were obtained for 102 of 105 (97%) of the maternal samples tested and all 25 of the infant samples tested. Complete double-stranded sequences were obtained for 90 of 105 (86%) of the maternal samples tested and 22 of 25 (88%) of the infant samples tested. The sequences obtained with this system were used for HIV-1 subtyping. The subtypes identified were A, C, D, and A/D recombinant HIV-1. The performances of the seven sequencing primers were similar for the subtypes examined. The ViroSeq system performs well for analysis of Ugandan plasma samples with subtypes A, C, D, and A/D recombinant HIV-1. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in countries where these subtypes are prevalent.
Prevalence of
Leishmania infantum
Infection in Dogs Living in an Area of Canine Leishmaniasis Endemicity Using PCR on Several Tissues and Serology
Authors: Solano-Gallego Laia , Morell Pere , Arboix Margarita , Alberola Jordi , Ferrer Lluis
Published: July 2002

We studied and compared the prevalence of
Leishmania
infection and the seroprevalence and the prevalence of canine leishmaniasis in an area where canine leishmaniasis is endemic. One hundred dogs living on the island of Mallorca (Spain) were studied. In this study, we clinically examined each dog for the presence of symptoms compatible with leishmaniasis, determined the titer of anti-
Leishmania
antibodies, and investigated the presence of
Leishmania
DNA by PCR in skin, conjunctiva, and bone marrow samples of each dog. The prevalence of the disease and the seroprevalence were 13 and 26%, respectively. In 63% of the dogs,
Leishmania
DNA could be detected by PCR in at least one of the tissues studied. The results of positive PCR in the bone marrow, the conjunctiva, and the skin were 17.8, 32, and 51%, respectively. The prevalence of the infection, 67%, was calculated using all animals that were seropositive and/or positive by PCR with any tissue. The results showed that the majority of dogs living in an area where canine leishmaniasis is endemic are infected by
Leishmania
and that the prevalence of infection is much greater than the prevalence of overt
Leishmania
-related disease.
Nocardia africana
sp. nov., a New Pathogen Isolated from Patients with Pulmonary Infections
Authors: Hamid Mohamed E. , Maldonado Luis , Sharaf Eldin Ghada S. , Mohamed Maha F. , Saeed Nageeb S. , Goodfellow Michael
Published: July 2002

Eight actinomycete strains, isolated from 8 out of 400 sputum samples examined, taken from patients with pulmonary diseases at the Chest Unit of Khartoum Teaching Hospital in the Sudan, were provisionally assigned to the genus
Nocardia
according to morphological criteria. These isolates were studied further in order to establish their taxonomic status. They were found to have morphological and chemical properties typical of nocardiae and formed a monophyletic clade in the 16S ribosomal DNA tree together with
Nocardia vaccinii
. The strains showed a unique pattern of phenotypic properties that distinguished them from representatives of recognized
Nocardia
species, including
Nocardia vaccinii
. The strains were considered to merit species status and were designated
Nocardia africana
sp. nov. The findings of the present study are consistent with the view that pulmonary nocardiosis may occur in a substantial proportion of patients who exhibit chronic lung diseases in African countries. It is important, therefore, that clinicians in such countries consider this condition, especially when patients with respiratory infections fail to respond to antitubercular therapy.
Identification of Canine Coronavirus Strains from Feces by S Gene Nested PCR and Molecular Characterization of a New Australian Isolate
Authors: Naylor Matthew J. , Harrison Gavan A. , Monckton Robert P. , McOrist Steven , Lehrbach Philip R. , Deane Elizabeth M.
Published: July 2002

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).
Diversity within Reference Strains of
Corynebacterium matruchotii
Includes
Corynebacterium durum
and a Novel Organism
Authors: Barrett Sara L. Rassoulian , Cookson Brad T. , Carlson LaDonna C. , Bernard Kathryn A. , Coyle Marie B.
Published: July 2002

Corynebacterium matruchotii
has been the subject of numerous dental pathogenesis studies. The purpose of the present study was to resolve concerns about diversity within the reference strains of
C. matruchotii
through analysis of seven strains procured from the American Type Culture Collection (ATCC). Analysis of whole-cell fatty acid profiles with the library generation software of Microbial ID Inc. revealed that three types of organisms have been deposited in the ATCC as
C. matruchotii
. These three groups of organisms were also distinguishable by DNA-DNA dot blot hybridization, by sequences of two hypervariable regions of the 16S rRNA gene, and by the pyrrolidonyl arylamidase test. These studies indicate that two
C. matruchotii
reference strains, ATCC 33449 and ATCC 33822, are members of the recently proposed species,
Corynebacterium durum
. The colonial morphology and biochemical reactions of the
C. durum
strains are more diverse than originally reported. Strain ATCC 43833 is unique and represents a novel species. In addition to the type strain, ATCC 14266, true members of the species
C. matruchotii
include ATCC strains 14265, 33806, and 43832 plus two reference strains, L2 and Richardson 13, which comprise the vast majority of strains used in dental pathogenesis research with this species.