bluuebunny/crossref_metadata_2025 huggingface dataset:
Extended Repertoire of Genes Encoding Variable Surface Lipoproteins in
Mycoplasma bovis
Strains
Authors: Nussbaum Sarit , Lysnyansky Inessa , Sachse Konrad , Levisohn Sharon , Yogev David
Published: July 2002

A genomic cluster of
vsp
genes was previously shown to mediate high-frequency phenotypic switching of surface lipoprotein antigens in the bovine pathogen
Mycoplasma bovis
. This study revealed that field strains of
M. bovis
possess modified versions of the
vsp
gene complex in which extensive sequence variations occur primarily in the reiterated coding sequences of the
vsp
structural genes. These findings demonstrate that there is a vastly expanded potential for antigenic variation within populations of this organism.
A Superoxide-HypersusceptibleSalmonella entericaSerovar Typhimurium Mutant Is Attenuated but Regains Virulence in p47phox−/−Mice
Authors: van Diepen Angela , van der Straaten Tahar , Holland Steven M. , Janssen Riny , van Dissel Jaap T.
Published: July 2002

Salmonella entericaserovar Typhimurium is a gram-negative, facultative intracellular pathogen that predominantly invades mononuclear phagocytes and is able to establish persistent infections. One of the innate defense mechanisms of phagocytic cells is the production of reactive oxygen species, including superoxide.S. entericaserovar Typhimurium has evolved mechanisms to resist such radicals, and these mechanisms could be decisive in its ability to survive and replicate within macrophages. Recently, we described a superoxide-hypersusceptibleS. entericaserovar Typhimurium mutant strain, DLG294, that carries a transposon insspJ, resulting in the lack of expression of SspJ, which is necessary for resistance against superoxide and replication within macrophages. Here we show that DLG294, which is a 14028s derivative, hardly induced any granulomatous lesions in the livers upon subcutaneous infection of C3H/HeN (Ityr) mice with 3 × 104bacteria and that its bacterial counts were reduced by 3 log units compared to those of wild-typeS. entericaserovar Typhimurium 14028s on day 5 after infection. In contrast, DLG294 replicated like wild-typeS. entericaserovar Typhimurium 14028s and induced a phenotypically similar liver pathology in p47phox−/−mice, which are deficient in the p47phoxsubunit of the NADPH oxidase complex and which do not produce superoxide. Consistent with these results, DLG294 reached bacterial counts identical to those of wild-typeS. entericaserovar Typhimurium 14028s in bone marrow-derived macrophages from p47phox−/−mice and in X-CGD PLB-985 cells at 24 h after challenge. These results indicate that SspJ plays a role in the bacterium's resistance to oxidative stress and in the survival and replication ofS. entericaserovar Typhimurium both in vitro and in vivo.
A Glutamine Transport Gene,
glnQ
, Is Required for Fibronectin Adherence and Virulence of Group B Streptococci
Authors: Tamura Glen S. , Nittayajarn Aphakorn , Schoentag Deborah L.
Published: July 2002

Group B streptococci (GBS) are a leading cause of neonatal sepsis and meningitis. GBS adhere to fibronectin when it is attached to a solid phase. We isolated a Tn
917
transposon mutant, COH1-GT1, which shows decreased adherence to fibronectin. COH1-GT1 also shows decreased adherence to and invasion of respiratory epithelial cells in vitro and decreased virulence in vivo. COH1-GT1 contains a Tn
917
insertion in a homolog of
glnQ
, a gene from
Escherichia coli
which is required for glutamine transport and codes for a cytoplasmic ATP-binding cassette protein. To confirm that the decreased fibronectin adherence of COH1-GT1 was due to the mutation in
glnQ
, we constructed COH1-GT2, a strain with a nonpolar site-directed mutation in
glnQ.
COH1-GT2 showed decreased binding to fibronectin. We also demonstrated that complementation of
glnQ
in
trans
restored fibronectin adherence to COH1-GT1. COH1-GT1 shows decreased uptake of radiolabeled glutamine and is resistant to the toxic glutamine analog γ-
l
-glutamylhydrazide, demonstrating that the
glnQ
gene is required for glutamine transport in GBS.
glnQ
lacks a signal sequence and is a cytoplasmic protein in
E. coli
and thus is unlikely to act as a fibronectin adhesin.
glnQ
is transcribed in an operon with a putative glutamine permease gene,
glnP
, which has a novel predicted structure containing three distinct domains linked in a single gene. The first two domains are putative glutamine binding domains with homology to the
E. coli
periplasmic glutamine binding gene
glnH
. The third is a putative permease domain with homology to the
E. coli
glutamine permease gene
glnP.
RT-PCR analysis demonstrated that
glnP
and
glnQ
are contained within a single transcript. Transcription of
scpB
, encoding the only known fibronectin-binding adhesin of GBS, is unaffected. We speculate that
glnQ
may regulate expression of fibronectin adhesins by affecting cytoplasmic glutamine levels and that regulation may be posttranscriptional.
Environmental Regulation of
Bacillus subtilis
ς
D
-Dependent Gene Expression
Authors: Mirel D. B. , Estacio W. F. , Mathieu M. , Olmsted E. , Ramirez J. , Márquez-Magaña L. M.
Published: July 2002

The ς
D
regulon of
Bacillus subtilis
is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of ς
D
protein levels and flagellin mRNA demonstrate that
sigD
expression and ς
D
activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the ς
D
-dependent flagellin gene (
hag
) as a model gene to study the effects of different nutritional environments on ς
D
-dependent gene expression. In complex medium, the level of expression of a
hag-lacZ
fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects
sigD
expression and/or ς
D
activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in
hag-lacZ
expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a
codY
mutant bearing a
hag-lacZ
reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes
abrB
,
hpr
, and
sinR
failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of
hag
nutritional repression in
B. subtilis.
Sequencing, Cloning, and High-Level Expression of the
pfp
Gene, Encoding a PP
i
-Dependent Phosphofructokinase from the Extremely Thermophilic Eubacterium
Dictyoglomus thermophilum
Authors: Ding Yan-Huai R. , Ronimus Ron S. , Morgan Hugh W.
Published: July 2002

The sequencing, cloning, and expression of the
pfp
gene from
Dictyoglomus thermophilum
, which consists of 1,041 bp and encodes a pyrophosphate-dependent phosphofructokinase, are described. A phylogenetic analysis indicates that the enzyme is closely related to the pyrophosphate-dependent enzyme from
Thermoproteus tenax
. The recombinant and native enzymes share a high degree of similarity for most properties examined.
Genetic Evidence that Transcription Activation by RhaS Involves Specific Amino Acid Contacts with Sigma 70
Authors: Bhende Prasanna M. , Egan Susan M.
Published: July 2002

RhaS activates transcription of the
Escherichia coli rhaBAD
and
rhaT
operons in response to
l
-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether ς
70
might be an activation target for RhaS. We found that ς
70
K593 and R599 appear to be important for RhaS activation at both
rhaBAD
and
rhaT
, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged ς
70
residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and ς
70
substitutions and found that RhaS D241 and ς
70
R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that ς
70
R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact ς
70
. More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with ς
70
may be conserved.
The Flagellar Filament of
Rhodobacter sphaeroides
: pH-Induced Polymorphic Transitions and Analysis of the
fliC
Gene
Authors: Shah Deepan S. H. , Perehinec Tania , Stevens Susan M. , Aizawa Shin-Ichi , Sockett R. Elizabeth
Published: July 2002

Flagellar motility in
Rhodobacter sphaeroides
is notably different from that in other bacteria.
R. sphaeroides
moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor.
R. sphaeroides
has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated
R. sphaeroides
filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the
R. sphaeroides
filament differs from that reported for
Salmonella enterica
serovar Typhimurium. The sequence of the
R. sphaeroides fliC
gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a ς
28
-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of
R. sphaeroides
flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.
Mutagenesis of
Neisseria meningitidis
by In Vitro Transposition of
Himar1 mariner
Authors: Pelicic Vladimir , Morelle Sandrine , Lampe David , Nassif Xavier
Published: July 2002

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

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

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

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

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

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

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

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

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

The 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

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

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

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.
Genetic Organization of the Region Encoding Regulation, Biosynthesis, and Transport of Rhizobactin 1021, a Siderophore Produced by
Sinorhizobium meliloti
Authors: Lynch Damien , O'Brien John , Welch Timothy , Clarke Paul , ÓCuı́v Páraic , Crosa Jorge H. , O'Connell Michael
Published: July 2002

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by
Sinorhizobium meliloti
. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named
rhbABCDEF
, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named
rhtA
, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the
rhbABCDEF
operon and the
rhtA
gene was found to be positively regulated by the product of the eighth gene in the cluster, named
rhrA
, which has characteristics of an AraC-type transcriptional activator. The six genes in the
rhbABCDEF
operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of
S. meliloti
2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for
rhbF
or
rhtA
at a time when the
nifH
gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.
Functional Characterization and Regulation ofgadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of theEscherichia coliGlutamic Acid Decarboxylase System
Authors: Tramonti Angela , Visca Paolo , De Canio Michele , Falconi Maurizio , De Biase Daniela
Published: July 2002

TheEscherichia colichromosome contains two distantly located genes,gadAandgadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H+and producing γ-aminobutyric acid. This compound is exported via the protein product of thegadCgene, which is cotranscribed withgadB. Here we demonstrate that transcription of bothgadAandgadBCis positively controlled bygadX, a gene downstream ofgadA, encoding a transcriptional regulator belonging to the AraC/XylS family. ThegadXpromoter encompasses the 67-bp region preceding thegadXtranscription start site and contains both RpoD and RpoS putative recognition sites. Transcription ofgadXoccurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of thegadstructural genes. However, PT5lacO-controlledgadXexpression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when thegadsystem is normally repressed. Autoregulation of the wholegadsystem is inferred by the positive effect of GadX on thegadApromoter andgadAXcotranscription. Transcription ofgadXis derepressed in anhnsmutant and strongly reduced in bothrpoSandhns rpoSmutants, consistent with the expression profile ofgadstructural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX bindsgadAandgadBCpromoters at different sites and with different binding affinities.
Elevated Levels of Ketopantoate Hydroxymethyltransferase (PanB) Lead to a Physiologically Significant Coenzyme A Elevation in
Salmonella enterica
Serovar Typhimurium
Authors: Rubio Aileen , Downs D. M.
Published: July 2002

Pantothenate is the product of the ATP-dependent condensation of pantoate and β-alanine and is a direct precursor of coenzyme A. A connection exists between pantothenate biosynthesis and thiamine biosynthesis in
Salmonella enterica
serovar Typhimurium since derivatives of a
purF
mutant that can grow (on glucose medium) in the absence of thiamine excrete pantothenate. We show here that the causative mutation in three such mutants was the addition of a CG base pair upstream of the
panB
gene. This base addition brings the spacing between the −10 and −35 hexamers of the promoter to a consensus spacing of 17 bp and results in increased transcription of the
pan
operon. Furthermore, overexpression of PanB caused by this mutation, or by other means, was necessary and sufficient to increase pantothenate production and allow PurF-independent thiamine synthesis on glucose medium.
Use of Plastic Vacutainer Tubes for Quantification of Human Immunodeficiency Virus Type 1 in Blood Specimens
Authors: Landry Marie L. , Garner Robin , Ferguson David
Published: July 2002

Human immunodeficiency virus type 1 viral load results were compared for paired samples collected in plastic and glass Vacutainer tubes, using both standard (
n
= 60) and ultrasensitive (
n
= 66) assays. The results showed a strong correlation (
P
< 0.0001), and plastic tubes can be substituted for glass tubes.
Use of Penicillin MICs To Predict In Vitro Activity of Other β-Lactam Antimicrobial Agents against
Streptococcus pneumoniae
Authors: Brueggemann A. B. , Pfaller M. A. , Doern G. V.
Published: July 2002

Linear regression analysis was used to compare penicillin MICs determined with 3,129 recent clinical isolates of
Streptococcus pneumoniae
to MICs obtained with nine other beta-lactam antimicrobial agents. A strong correlation between penicillin MICs and those obtained with other beta-lactams was demonstrated. It may be possible to test penicillin and use MICs obtained with penicillin to predict MICs of other beta-lactam antimicrobials for
Streptococcus pneumoniae
.
Skin Disease Presenting as an Outbreak of Pseudobacteremia in a Laboratory Worker
Authors: Simhon A. , Rahav G. , Shapiro M. , Block C.
Published: July 2002

An outbreak of pseudobacteremia due to
Streptococcus pyogenes
(group A streptococci [GAS]) and methicillin-susceptible
Staphylococcus aureus
(MSSA) was traced to the venting procedure for aerobic bottles prior to their loading into the incubator of the BacT/Alert analyzer (Organon Teknika). Bacteria shed by a laboratory worker suffering from impetigo and cellulitis contaminated the aerobic bottles of 10 patients. All blood culture isolates, in addition to the isolates from the laboratory worker, were of the same GAS M and T types. All MSSA isolates from blood cultures and the index case's hands had the same lytic phage profile. Procedural breakdowns were identified in the laboratory. Bottles were vented outside the biological safety cabinet, gloves were not worn, and unprotected needles were used for the venting procedure. The source of the aspirated bacteria that contaminated the bottles was identified and the index case was treated promptly.
Influence of Variations in Test Methods on Susceptibility of
Haemophilus influenzae
to Ampicillin, Azithromycin, Clarithromycin, and Telithromycin
Authors: Fuchs Peter C. , Barry Arthur L. , Brown Steven D.
Published: July 2002

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of
Haemophilus influenzae
to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO
2
most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for β-lactamase-negative strains from 1.50 to 2.45 μg/ml. In addition, 206
H. influenzae
strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with
Haemophilus
test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against
H. influenzae
.
Comparison of the Vitek Gram-Positive Susceptibility 106 Card, the MRSA-Screen Latex Agglutination Test, and
mecA
Analysis for Detecting Oxacillin Resistance in a Geographically Diverse Collection of Clinical Isolates of Coagulase-Negative Staphylococci
Authors: Yamazumi T. , Furuta I. , Diekema D. J. , Pfaller M. A. , Jones R. N.
Published: July 2002

The Vitek automated susceptibility testing system with a modified gram-positive susceptibility (GPS) 106 card (bioMerieux Vitek, Inc., Hazelwood. Mo.) and a rapid slide latex agglutination test (MRSA-Screen test; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their abilities to detect oxacillin resistance in coagulase-negative staphylococci (CoNS). The reference broth microdilution method and the detection of the
mecA
gene by PCR (“gold standard” reference result) were used to compare the results obtained with the commercial products. A total of 123 clinical isolates consisting of eight species were selected from U.S. surveillance collections. Among the
mecA
-positive isolates (95 strains), 30 isolates were initially negative on the MRSA-Screen test read at 3 min. When the agglutination reaction was extended for 10 min, 26 of the 30 isolates became positive. For a different four isolates, the oxacillin MIC was ≤0.25 μg/ml on the Vitek GPS 106 card. Among the
mecA
-negative isolates (28 strains), for two
Staphylococcus warneri
, two
S. lugdunensis
, and two
S. saprophyticus
strains MICs were ≥0.5 μg/ml by the reference broth microdilution method. Four of these isolates were also categorized as resistant with the Vitek GPS 106 card and two isolates were positive by the MRSA-Screen test. Overall, the MRSA-Screen test, GPS 106 card, and reference broth microdilution method had sensitivities of 95.7 (result at 10 min), 95.7, and 100%, respectively, and specificities of 92.8, 85.7, and 78.5%, respectively. Although the MRSA-Screen test required a slight procedural modification, both commercial methods achieved a sensitivity and specificity at detecting oxacillin resistance in CoNS at a level that was acceptable for clinical laboratory use.
Identification of
Staphylococcus
spp. by PCR-Restriction Fragment Length Polymorphism of
gap
Gene
Authors: Yugueros Javier , Temprano Alejandro , Sánchez Marı́a , Luengo José Marı́a , Naharro Germán
Published: July 2002

Oligonucleotide primers specific for the
Staphylococcus aureus gap
gene were previously designed to identify 12
Staphylococcus
spp. by PCR. In the present study,
Alu
I digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24
Staphylococcus
spp. to be identified with high specificity.
Contamination of the Clinical Microbiology Laboratory with Vancomycin-Resistant Enterococci and Multidrug- Resistant
Enterobacteriaceae
: Implications for Hospital and Laboratory Workers
Authors: Collins Susan M. , Hacek Donna M. , Degen Lisa A. , Wright Marc O. , Noskin Gary A. , Peterson Lance R.
Published: July 2002

We surveyed environmental surfaces in our clinical microbiology laboratory to determine the prevalence of vancomycin-resistant enterococci (VRE) and multidrug-resistant
Enterobacteriaceae
(MDRE) during a routine working day. From a total of 193 surfaces, VRE were present on 20 (10%) and MDRE were present on 4 (2%) of the surfaces tested. In a subsequent survey after routine cleaning, all of the 24 prior positive surfaces were found to be negative. Thus, those in the laboratory should recognize that many surfaces may be contaminated by resistant organisms during routine processing of patient specimens.
PCR Analyses of tRNA Intergenic Spacer, 16S-23S Internal Transcribed Spacer, and Randomly Amplified Polymorphic DNA Reveal Inter- and Intraspecific Relationships of
Enterobacter cloacae
Strains
Authors: Clementino Maysa M. , de Filippis Ivano , Nascimento Carlos R. , Branquinho Regina , Rocha Carmem L. , Martins Orlando B.
Published: July 2002

PCR analysis of tRNA intergenic spacer (tDNA-PCR) and of the 16S-23S internal transcribed spacer (ITS-PCR) and random amplified polymorphic DNA (RAPD) analysis were evaluated for their usefulness in characterization of
Enterobacter cloacae
strains isolated from both clinical origins and vaccine microbial contamination. tDNA-PCR presented specific and reproducible patterns for
Enterobacter sakazakii
ATCC 29004,
Enterobacter aerogenes
ATCC 13048, and
Enterobacter cloacae
ATCC 13047 and 23355 that presented the same profile for all 16
E. cloacae
isolates, offering an alternative tool for species-level identification. ITS-PCR and RAPD analysis yielded completely different banding patterns for the 20 strains studied, except for
E. cloacae
strains isolated from different batches of vaccine that exhibited a unique pattern, suggesting contamination by the same strain. The combined use of tDNA-PCR and ITS-PCR in a one-step protocol allows accurate identification and typing of
E. cloacae
strains a few hours after the colony has been isolated.