bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Forced Convection on the Skin Thermal Expression of Breast Cancer
Authors: Hu Lu , Gupta Ashish , Gore Jay P. , Xu Lisa X.
Published: May 2004

A bioheat-transfer-based numerical model was utilized to study the energy balance in healthy and malignant breasts subjected to forced convection in a wind tunnel. Steady-state temperature distributions on the skin surface of the breasts were obtained by numerically solving the conjugate heat transfer problem. Parametric studies on the influences of the airflow on the skin thermal expression of tumors were performed. It was found that the presence of tumor may not be clearly shown due to the irregularities of the skin temperature distribution induced by the airflow field. Nevertheless, image subtraction techniques could be employed to eliminate the effects of the flow field and thermal noise and significantly improve the thermal signature of the tumor on the skin surface. Inclusion of the possible skin vascular response to cold stress caused by the airflow further enhances the signal, especially for deeply embedded tumors that otherwise may not be detectable.
Effect of Fluid Boundary Conditions on Joint Contact Mechanics and Applications to the Modeling of Osteoarthritic Joints
Authors: Federico Salvatore , La Rosa Guido , Herzog Walter , Wu John Z.
Published: May 2004

The long-term goal of our research is to understand the mechanism of osteoarthritis (OA) initiation and progress through experimental and theoretical approaches. In previous theoretical models, joint contact mechanics was implemented without consideration of the fluid boundary conditions and with constant permeability. The primary purpose of this study was to investigate the effect of fluid boundary conditions at the articular surfaces on the contact mechanics, in terms of load sharing and fluid flow properties using variable permeability. The tested conditions included totally sealed surfaces, open surfaces, and open surfaces with variable permeability. While the sealed surface model failed to predict relaxation times and load sharing properly, the class of open surface models (open surfaces with constant permeability, and surfaces with variable permeability) gave good agreement with experiments, in terms of relaxation time and load sharing between the solid and the fluid phase. In particular, the variable permeability model was judged to be the most realistic of the three models, from a biological and physical point of view. This model was then used to simulate joint contact in the early and late stages of OA. In the early stages of OA, the model predicted a decrease in peak contact pressure and an increase in contact area, while in the late stages of OA, peak pressures were increased and contact areas were decreased compared to normal. These findings agree well with experimental observations.
A Proposed Injury Threshold for Mild Traumatic Brain Injury
Authors: Zhang Liying , Yang King H. , King Albert I.
Published: May 2004

Traumatic brain injuries constitute a significant portion of injury resulting from automotive collisions, motorcycle crashes, and sports collisions. Brain injuries not only represent a serious trauma for those involved but also place an enormous burden on society, often exacting a heavy economical, social, and emotional price. Development of intervention strategies to prevent or minimize these injuries requires a complete understanding of injury mechanisms, response and tolerance level. In this study, an attempt is made to delineate actual injury causation and establish a meaningful injury criterion through the use of the actual field accident data. Twenty-four head-to-head field collisions that occurred in professional football games were duplicated using a validated finite element human head model. The injury predictors and injury levels were analyzed based on resulting brain tissue responses and were correlated with the site and occurrence of mild traumatic brain injury (MTBI). Predictions indicated that the shear stress around the brainstem region could be an injury predictor for concussion. Statistical analyses were performed to establish the new brain injury tolerance level.
Mechanical Thresholds for Initiation and Persistence of Pain Following Nerve Root Injury: Mechanical and Chemical Contributions at Injury
Authors: Winkelstein Beth A. , DeLeo Joyce A.
Published: May 2004

There is much evidence supporting the hypothesis that magnitude of nerve root mechanical injury affects the nature of the physiological responses which can contribute to pain in lumbar radiculopathy. Specifically, injury magnitude has been shown to modulate behavioral hypersensitivity responses in animal models of radiculopathy. However, no study has determined the mechanical deformation thresholds for initiation and maintenance of the behavioral sensitivity in these models. Therefore, it was the purpose of this study to quantify the effects of mechanical and chemical contributions at injury on behavioral outcomes and to determine mechanical thresholds for pain onset and persistence. Male Holtzman rats received either a silk or chromic gut ligation of the L5 nerve roots, a sham exposure of the nerve roots, or a chromic exposure in which no mechanical deformation was applied but chromic gut material was placed on the roots. Using image analysis, nerve root radial strains were estimated at the time of injury. Behavioral hypersensitivity was assessed by measuring mechanical allodynia continuously throughout the study. Chromic gut ligations produced allodynia responses for nerve root strains at two-thirds of the magnitudes of those strains which produced the corresponding behaviors for silk ligation. Thresholds for nerve root compression producing the onset (8.4%) and persistence of pain (17.4%–22.2%) were determined for silk ligation in this lumbar radiculopathy model. Such mechanical thresholds for behavioral sensitivity in a painful radiculopathy model begin to provide biomechanical data which may have utility in broader experimental and computational models for relating injury biomechanics and physiologic responses of pain.
On the Effects of Residual Stress in Microindentation Tests of Soft Tissue Structures
Authors: Zamir Evan A. , Taber Larry A.
Published: May 2004

Microindentation methods are commonly used to determine material properties of soft tissues at the cell or even sub-cellular level. In determining properties from force-displacement (FD) data, it is often assumed that the tissue is initially a stress-free, homogeneous, linear elastic half-space. Residual stress, however, can strongly influence such results. In this paper, we present a new microindentation method for determining both elastic properties and residual stress in soft tissues that, to a first approximation, can be regarded as a pre-stressed layer embedded in or adhered to an underlying relatively soft, elastic foundation. The effects of residual stress are shown using two linear elastic models that approximate specific biological structures. The first model is an axially loaded beam on a relatively soft, elastic foundation (i.e., stress-fiber embedded in cytoplasm), while the second is a radially loaded plate on a foundation (e.g., cell membrane or epithelium). To illustrate our method, we use a nonlinear finite element (FE) model and experimental FD and surface contour data to find elastic properties and residual stress in the early embryonic chick heart, which, in the region near the indenter tip, is approximated as an isotropic circular plate under tension on a foundation. It is shown that the deformation of the surface in a microindentation test can be used along with FD data to estimate material properties, as well as residual stress, in soft tissue structures that can be regarded as a plate under tension on an elastic foundation. This method may not be as useful, however, for structures that behave as a beam on a foundation.
In Vivo Measurement of the Dynamic 3-D Kinematics of the Ovine Stifle Joint
Authors: Tapper, Janet E. , Ronsky, Professor Janet L. , Powers, Ph.D. Candidate Marilyn J. , Sutherland, Animal Care Technician Craig , Majima, Research Fellow Tokifumi , Frank, Professor & Division Head, Orthopaedic Surgery Cyril B. , Shrive, Professor Nigel G.
Published: May 2004

The ovine stifle joint is a promising animal model for investigation of joint mechanobiology. A method for in vivo measurement of dynamic 3-D kinematics of the ovine stifle joint is described (accuracy: 0.36±0.39 mm). Inter-subject variability in kinematics is greater than both intra-subject and inter-session variability. For future studies in which joint kinematics are measured prior to and following controlled orthopaedic interventions, pooling of data should be avoided and each subject should act as its own control.
Infrared Signature of Structures Associated with the H
+
(H
2
O)

n

(
n
= 6 to 27) Clusters
Authors: Shin J.-W. , Hammer N. I. , Diken E. G. , Johnson M. A. , Walters R. S. , Jaeger T. D. , Duncan M. A. , Christie R. A. , Jordan K. D.
Published: May 2004

We report the OH stretching vibrational spectra of size-selected H
+
(H
2
O)

n

clusters through the region of the pronounced “magic number” at
n
= 21 in the cluster distribution. Sharp features are observed in the spectra and assigned to excitation of the dangling OH groups throughout the size range 6 ≤
n
≤ 27. A multiplet of such bands appears at small cluster sizes. This pattern simplifies to a doublet at
n
= 11, with the doublet persisting up to
n
= 20, but then collapsing to a single line in the
n
= 21 and
n
= 22 clusters and reemerging at
n
= 23. This spectral simplification provides direct evidence that, for the magic number cluster, all the dangling OH groups arise from water molecules in similar binding sites.
OmpR and LeuO Positively Regulate the
Salmonella enterica
Serovar Typhi
ompS2
Porin Gene
Authors: Fernández-Mora Marcos , Puente José Luis , Calva Edmundo
Published: May 2004

The
Salmonella enterica
serovar Typhi
ompS2
gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for
S. enterica
serovar Typhi OmpS1,
Escherichia coli
OmpN, and
Klebsiella pneumoniae
OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in
ompS2
expression in an
S. enterica
serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory
ompB
(
ompR envZ
) operon. Furthermore,
ompS2
expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of
ompS2
expression. Augmented expression of
ompS2
was also obtained upon addition of cloned
leuO
to the wild-type strain, but not in an
ompR
isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate
ompS2
.
Functional Dissection of the XpsN (GspC) Protein of the
Xanthomonas campestris
pv. campestris Type II Secretion Machinery
Authors: Lee Hsien-Min , Chen Juine-Ruey , Lee Hai-Lun , Leu Wei-Ming , Chen Ling-Yun , Hu Nien-Tai
Published: May 2004

Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen
Xanthomonas campestris
pv. campestris. All are bitopic cytoplasmic-membrane proteins, each with a large C-terminal periplasmic domain. They have been demonstrated to form a dissociable ternary complex. By analyzing the C-terminally truncated XpsN and PhoA fusions, we discovered that truncation of the C-terminal 103 residues produced a functional protein, albeit present below detectable levels. Furthermore, just the first 46 residues, encompassing the membrane-spanning sequence (residues 10 to 32), are sufficient to keep XpsL and XpsM at normal abundance. XpsN46(His
6
), synthesized in
Escherichia coli
, is able to associate in a membrane-mixing experiment with the XpsL-XpsM complex preassembled in
X. campestris
pv. campestris. The XpsN N-terminal 46 residues are apparently sufficient not only for maintaining XpsL and XpsM at normal levels but also for their stable association. The membrane-spanning sequence of XpsN was not replaceable by that of TetA. However, coimmunoprecipitation with XpsL and XpsM was observed for XpsN97::PhoA, but not XpsN46::PhoA. Only XpsN97::PhoA is dominant negative. Single alanine substitutions for three charged residues within the region between residues 47 and 97 made the protein nonfunctional. In addition, the R78A mutant XpsN protein was pulled down by XpsL-XpsM(His
6
) immobilized on an Ni-nitrilotriacetic acid column to a lesser extent than the wild-type XpsN. Therefore, in addition to the N-terminal 46 residues, the region between residues 47 and 97 of XpsN probably also plays an important role in interaction with XpsL-XpsM.
TcaR, a Putative MarR-Like Regulator of
sarS
Expression
Authors: McCallum Nadine , Bischoff Markus , Maki Hideki , Wada Akihito , Berger-Bächi Brigitte
Published: May 2004

TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel
Staphylococcus aureus
regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates
sarS
and thus
spa
transcription. In addition, it attenuates whole-length transcription of
sasF
, thereby producing a truncated transcript lacking the 3′ terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact
tcaR
gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of
tcaR
, harboring an inactive TcaR and therefore expressing very low levels of
sarS
. The data presented here identified TcaR as a further activator of
sarS
, and a modulator of
sasF
expression that has to be taken into account in studies of virulence gene expression in
S. aureus
.
Assembly of the MexAB-OprM Multidrug Efflux System of
Pseudomonas aeruginosa
: Identification and Characterization of Mutations in
mexA
Compromising MexA Multimerization and Interaction with MexB
Authors: Nehme Dominic , Li Xian-Zhi , Elliot Rachel , Poole Keith
Published: May 2004

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of
P. aeruginosa
is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of
Escherichia coli
, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.
Identification of
rhtX
and
fptX
, Novel Genes Encoding Proteins That Show Homology and Function in the Utilization of the Siderophores Rhizobactin 1021 by
Sinorhizobium meliloti
and Pyochelin by
Pseudomonas aeruginosa
, Respectively
Authors: Ó Cuív Páraic , Clarke Paul , Lynch Damien , O'Connell Michael
Published: May 2004

Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium
Sinorhizobium meliloti
2011. A regulon comprising
rhtA
, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon
rhbABCDEF
; and
rhrA
, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of
rhbA
and named
rhtX
(for “rhizobactin transport”), which is required, in addition to
rhtA
, to confer the ability to utilize rhizobactin 1021 on a strain of
S. meliloti
that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in
Escherichia coli
via the IutA outer membrane receptor and the FhuCDB inner membrane transport system.
E. coli
expressing
iutA
and
fhuCDB
was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in
E. coli
. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of
Pseudomonas aeruginosa
, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named
fptX
(for “ferripyochelin transport”). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.
Transcription of the
Staphylococcus aureus cid
and
lrg
Murein Hydrolase Regulators Is Affected by Sigma Factor B
Authors: Rice Kelly C. , Patton Toni , Yang Soo-Jin , Dumoulin Alexis , Bischoff Markus , Bayles Kenneth W.
Published: May 2004

The
Staphylococcus aureus lrg
and
cid
loci are homologous operons that have been shown to regulate murein hydrolase activity and affect sensitivity to penicillin. Although the mode of action of these operons has not been demonstrated, a model based on the similarities of the
lrgA
and
cidA
gene products to the bacteriophage holin family of proteins has been proposed. In this study, the transcription organization and regulation of these operons were examined by Northern blot analyses. Unexpectedly,
cidB
and a gene located immediately downstream, designated
cidC
, were found to be cotranscribed on a 2.7-kb transcript. Maximal
cidBC
transcription occurred during early exponential growth, and high-level transcription of
cidBC
was dependent on the
rsbU
-mediated activation of the alternative sigma factor B (σ
B
). In contrast,
lrgAB
transcription in stationary phase was negatively regulated by σ
B
. Although
cidABC
transcription was not detected by Northern blot analysis, reverse transcriptase PCR revealed that these genes are also cotranscribed as a single RNA message in early exponential growth. Primer extension analysis revealed the presence of two
cidBC
transcription start sites, but no apparent σ
B
-dependent promoter consensus sequence was identified in these regions. The
rsbU
gene was also shown to have a positive impact on murein hydrolase activity but a negligible effect on sensitivity to penicillin-induced killing. These results suggest that the
lrgAB
and
cidBC
genes may be part of the
S. aureus
σ
B
-controlled stress regulon.
Expression of
magA
in
Legionella pneumophila
Philadelphia-1 Is Developmentally Regulated and a Marker of Formation of Mature Intracellular Forms
Authors: Hiltz Margot F. , Sisson Gary R. , Brassinga Ann Karen C. , Garduno Elizabeth , Garduno Rafael A. , Hoffman Paul S.
Published: May 2004

Legionella pneumophila
displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs). Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as “Mip-like” protein, that was enriched in MIFs. However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of
L. pneumophila
, and for clarity we renamed it MagA (for “MIF-associated gene”). We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (∼3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells. DNA sequence analysis of the
magA
locus revealed an upstream divergently transcribed gene,
msrA
, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as −10 hexamers often associated with stationary-phase regulation. While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress. MIFs from
L. pneumophila
strain Lp02 containing a
magA
deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits. As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development.
Temperature Sensitivity Caused by Mutant Release Factor 1 Is Suppressed by Mutations That Affect 16S rRNA Maturation
Authors: Kaczanowska Magdalena , Rydén-Aulin Monica
Published: May 2004

To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the
rplM-rpsI
operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the
yrdC
open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1.
Bacillus subtilisSalA (YbaL) Negatively Regulates Expression ofscoC, Which Encodes the Repressor for the Alkaline Exoprotease Gene,aprE
Authors: Ogura Mitsuo , Matsuzawa Atsushi , Yoshikawa Hirofumi , Tanaka Teruo
Published: May 2004

During the course of screening for exoprotease-deficient mutants amongBacillus subtilisgene disruptants, a strain showing such a phenotype was identified. The locus responsible for this phenotype was the previously unknown geneybaL, which we renamedsalA.The predicted gene product encoded bysalAbelongs to the Mrp family, which is widely conserved among archaea, prokaryotes, and eukaryotes. Disruption ofsalAresulted in a decrease in the expression of alacZfusion of theaprEgene encoding the major extracellular alkaline protease. The decrease was recovered by the clonedsalAgene on a plasmid, demonstrating that the gene is involved inaprEexpression. Determination of thecis-acting region of SalA on the upstream region ofaprE, together with epistatic analyses withscoC,abrB, andspo0Amutations that also affectaprEexpression, suggested thatsalAdeficiency affectsaprE-lacZexpression through the negative regulator ScoC. Northern and reverse transcription-PCR analyses revealed enhanced levels ofscoCtranscripts in thesalAmutant cells in the transition and early stationary phases. Concomitant with these observations, larger amounts of the ScoC protein were detected in the mutant cells by Western analysis. From these results we conclude that SalA negatively regulatesscoCexpression. It was also found that the expression of asalA-lacZfusion was increased bysalAdeficiency, suggesting thatsalAis autoregulated.
Instability of Pathogenicity Islands in Uropathogenic
Escherichia coli
536
Authors: Middendorf Barbara , Hochhut Bianca , Leipold Kristina , Dobrindt Ulrich , Blum-Oehler Gabriele , Hacker Jörg
Published: May 2004

The uropathogenic
Escherichia coli
strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of
E. coli
536 with the counterselectable marker
sacB
to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II
536
and PAI III
536
were the most unstable islands followed by PAI I
536
and PAI V
536
, whereas PAI IV
536
was stable. In addition, we found that deletion of PAI II
536
and PAI III
536
was induced by several environmental stimuli. Whereas excision of PAI I
536
, PAI II
536
, and PAI V
536
was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III
536
was deleted either by site-specific recombination or by homologous recombination between two IS
100
-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II
536
and PAI III
536
were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic
E. coli
can be modulated by deletion of PAIs.
Distinctive Protein Signatures Provide Molecular Markers and Evidence for the Monophyletic Nature of the
Deinococcus-Thermus
Phylum
Authors: Griffiths Emma , Gupta Radhey S.
Published: May 2004

The
Deinococcus-Thermus
group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees. No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the
Deinococcus-Thermus
phylum but are not found in any other group of bacteria. The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase β′ subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (σ
70
), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs. By using PCR primers for conserved regions, fragments of these genes were amplified from a number of
Deinococcus-Thermus
species, and all such fragments (except SerRS in
Deinococcus proteolyticus
) were found to contain the indicated signatures. The presence of these signatures in various species from all three known genera within this phylum, viz.,
Deinococcus
,
Thermus
, and
Meiothermus
, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group. The signature in SerRS, which is absent in
D. proteolyticus
, was likely introduced after the branching of this species. Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., σ
70
and SerRS) also support a sister group relationship between the
Thermus
and the
Meiothermus
genera. The identified signatures provide strong evidence for the monophyletic nature of the
Deinococcus-Thermus
phylum. These molecular markers should prove very useful in the identification of new species related to this group.
An Extracytoplasmic-Function Sigma Factor Is Involved in a Pathway Controlling β-Exotoxin I Production in
Bacillus thuringiensis
subsp.
thuringiensis
Strain 407-1
Authors: Espinasse Sylvain , Gohar Michel , Lereclus Didier , Sanchis Vincent
Published: May 2004

β-Exotoxin I is an insecticidal nucleotide analogue secreted by various
Bacillus thuringiensis
strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated
sigW
-
ecfX
-
ecfY
, that is essential for β-exotoxin I production in
B. thuringiensis
subsp.
thuringiensis
strain 407-1. In this strain, the disruption of the
sigW
cluster resulted in nontoxic culture supernatants.
sigW
encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from
Bacillus anthracis
strain Ames and
Bacillus cereus
strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from
Clostridium perfringens
and SigW and SigX from
Bacillus subtilis
, respectively. EcfX, encoded by the gene following
sigW
, significantly repressed the expression of
sigW
when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable
cry
plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1(Cry
−
)(Pig
+
), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of
sigW
indicated that this gene was expressed during the stationary phase and only in the 407-1(Cry
−
)(Pig
+
) mutant. This suggests that in the wild type-407(Cry
+
) strain, β-exotoxin I was produced from determinants located on a
cry
gene-bearing plasmid and that
sigW
is able to induce β-exotoxin I production in
B. thuringiensis
in the absence of
cry
gene
-
bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that β-exotoxin I production in
B. thuringiensis
can be restored or induced via an alternative pathway that requires
sigW
expression.
Toluene 3-Monooxygenase of
Ralstonia pickettii
PKO1 Is a
para
-Hydroxylating Enzyme
Authors: Fishman Ayelet , Tao Ying , Wood Thomas K.
Published: May 2004

Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of
Pseudomonas mendocina
KR1 hydroxylates monosubstituted benzenes at the
para
position and toluene
ortho
-monooxygenase (TOM) of
Burkholderia cepacia
G4 hydroxylates at the
ortho
position, toluene 3-monooxygenase (T3MO) of
Ralstonia pickettii
PKO1 was reported previously to hydroxylate toluene at the
meta
position, producing primarily
m
-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the
para
position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent

K
m

value of 250 μM and produced 90%
p
-cresol and 10%
m
-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97%
p
-cresol and 3%
m
-cresol.
Pseudomonas aeruginosa
PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66%
p
-nitrophenol and 34%
m
-nitrophenol from nitrobenzene and 100%
p
-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the
tbu
locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors.
1
H nuclear magnetic resonance analysis confirmed the structural identity of
p
-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
Characterization of Monospecies Biofilm Formation by
Helicobacter pylori
Authors: Cole Sheri P. , Harwood Julia , Lee Richard , She Rosemary , Guiney Donald G.
Published: May 2004

As all bacteria studied to date, the gastric pathogen
Helicobacter pylori
has an alternate lifestyle as a biofilm.
H. pylori
forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral
H. pylori
to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed
H. pylori
arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the
H. pylori
life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic
H. pylori
while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach,
H. pylori
planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene,
luxS
, and the
cagE
type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by
H. pylori
and the selective enhancement of planktonic growth in the presence of gastric mucin.
Generation of Enhanced Competitive Root-Tip-Colonizing
Pseudomonas
Bacteria through Accelerated Evolution
Authors: de Weert Sandra , Dekkers Linda C. , Kuiper Irene , Bloemberg Guido V. , Lugtenberg Ben J. J.
Published: May 2004

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn
5luxAB
mutant bank of the good root colonizer
Pseudomonas fluorescens
WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn
5luxAB
flanking regions showed that the transposon had inserted in a
mutY
homolog. This gene is involved in the repair of A · G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another
mutY
mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new
mutY
mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the
mutY
mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.
Organization and Expression of the Polynucleotide Phosphorylase Gene (
pnp
) of
Streptomyces
: Processing of
pnp
Transcripts in
Streptomyces antibioticus
Authors: Bralley Patricia , Jones George H.
Published: May 2004

We have examined the expression of
pnp
encoding the 3′-5′-exoribonuclease, polynucleotide phosphorylase, in
Streptomyces antibioticus
. We show that the
rpsO
-
pnp
operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both
pnp
and the gene for ribosomal protein S15 (
rpsO
) and a second, P
pnp
, located in the
rpsO-pnp
intergenic region. Unlike the situation in
Escherichia coli
, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type
S. antibioticus
mycelia. The P
pnp
transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5′ ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The P
pnp
sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in
rspO-pnp
structure and function between
S. antibioticus
and
E. coli
. In
E. coli
,
pnp
autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an
rpsO-pnp
intergenic hairpin. Computer modeling of the secondary structure of the
S. antibioticus
readthrough transcript predicts a stem-loop structure analogous to that in
E. coli
. However, our analysis suggests that while the readthrough transcript observed in
S. antibioticus
may be processed by an RNase III-like activity, transcripts originating from P
pnp
are not. Furthermore, the
S. antibioticus rpsO-pnp
intergenic region contains two open reading frames. The larger of these,
orfA
, may be a pseudogene. The smaller open reading frame,
orfX
, also observed in
Streptomyces coelicolor
and
Streptomyces avermitilis
, may be translationally coupled to
pnp
and the gene downstream from
pnp
, a putative protease.
Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the
Escherichia coli
Flagellar Motor
Authors: Van Way Susan M. , Millas Stephanos G. , Lee Aaron H. , Manson Michael D.
Published: May 2004

The FliG protein is a central component of the bacterial flagellar motor. It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor. FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa. A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB. We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker. Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused. Isolation of these mutants was facilitated by a “sensitizing” mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal
fliG
gene into our vector plasmid. This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence. The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from
Thermotoga maritima
.
Complexity of Gas Vesicle Biogenesis inHalobacteriumsp. Strain NRC-1: Identification of Five New Proteins
Authors: Shukla Hem Dutt , DasSarma Shiladitya
Published: May 2004

The genome ofHalobacteriumsp. strain NRC-1 contains a large gene cluster,gvpMLKJIHGFEDACNO, that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additionalgvpgene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-typeHalobacteriumsp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and β-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.
Arsenic Resistance inHalobacteriumsp. Strain NRC-1 Examined by Using an Improved Gene Knockout System
Authors: Wang Gejiao , Kennedy Sean P. , Fasiludeen Sabeena , Rensing Christopher , DasSarma Shiladitya
Published: May 2004

The genome sequence ofHalobacteriumsp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRCandarsR2M) and on the chromosome (arsB). We studied the role of thesearsgenes in arsenic resistance genetically by construction of gene knockouts. Deletion of thearsADRCgene cluster in aHalobacteriumNRC-1 Δura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomalarsBgene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of thearsMgene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate thatHalobacteriumsp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements inHalobacteriumthat relies on both selection and counterselection ofura3using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed thatarsA,arsD,arsR,arsM,arsC, andarsB, but notarsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.
Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of
Bacillus subtilis
by Inclusion in a Model Membrane
Authors: Searls Tim , Chen Xingyong , Allen Stephanie , Yudkin Michael D.
Published: May 2004

Starvation induces
Bacillus subtilis
to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor σ
F
, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of
B. subtilis
precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.
Salmonella enterica
Serovar Typhi Strains from Which SPI7, a 134-Kilobase Island with Genes for Vi Exopolysaccharide and Other Functions, Has Been Deleted
Authors: Nair Satheesh , Alokam Suneetha , Kothapalli Sushma , Porwollik Steffan , Proctor Emily , Choy Carmen , McClelland Michael , Liu Shu-Lin , Sanderson Kenneth E.
Published: May 2004

Salmonella enterica
serovar Typhi has a 134-kb island of DNA identified as salmonella pathogenicity island 7 (SPI7), inserted between
pheU
and ′
pheU
(truncated), two genes for tRNA
Phe
. SPI7 has genes for Vi exopolysaccharide, for type IVB pili, for putative conjugal transfer, and for sopE bacteriophage. Pulsed-field gel electrophoresis following digestion with the endonuclease I-CeuI, using DNA from a set of 120 wild-type strains of serovar Typhi assembled from several sources, identified eight strains in which the I-CeuI G fragment, which contains SPI7, had a large deletion. In addition, agglutination tests with Vi antiserum and phage typing with Vi phages show that all eight strains are Vi negative. We therefore tested these strains for deletion of SPI7 by multiplex PCR, by microarray analysis, and by sequencing of PCR amplicons. Data show that seven of the eight strains are precise deletions of SPI7: a primer pair flanking SPI7 results in a PCR amplicon containing a single
pheU
gene; microarrays show that all SPI7 genes are deleted. Two of the strains produce amplicons which have A derived from
pheU
at bp 27, while five have C derived from ′
pheU
at this position; thus, the position of the crossover which results in the deletion can be inferred. The deletion in the eighth strain, TYT1669, removes 175 kb with junction points in genes STY4465 and STY4664; the left junction of SPI7 and adjacent genes, as well as part of SPI7 including the
viaB
operon for Vi exopolysaccharide, was removed, while the right junction of SPI7 was retained. We propose that these deletions occurred during storage following isolation.