bluuebunny/crossref_metadata_2025 huggingface dataset:
Spinal Dorsal Horn Cell Receptive Field Size is Increased in Adult Rats Following Neonatal Hindpaw Skin Injury
Authors: Torsney Carole , Fitzgerald Maria
Published: May 2003

Local tissue damage in newborn rats can lead to changes in skin sensitivity that last into adulthood and this is likely to be due to plasticity of developing peripheral and central sensory connections. This study examines the functional connections of dorsal horn neurons in young and adult rats that have undergone local skin damage at birth. Newborn rat pups were halothane anaesthetised and received either a unilateral subcutaneous plantar injection of 1 % λ‐carrageenan or a unilateral plantar foot injury made by removal of 2 mm × 2 mm of skin. At 3 weeks, (postnatal day (P) 19–23) and 6 weeks (P40–44) in vivo extracellular recordings of single dorsal horn cells with plantar cutaneous receptive fields were made under urethane anaesthesia (2 g kg−1) and responses to mechanical and electrical stimulation of the skin were assessed. Following neonatal carrageenan inflammation, dorsal horn neuron properties and receptive field sizes at 3 weeks were the same as those of controls. In contrast, following neonatal skin injury, dorsal horn cell receptive field sizes were significantly greater than those of controls at 3 weeks (2.5‐fold) and at 6 weeks (2.2‐fold). Mechanical thresholds, mechanical response magnitudes and evoked responses to single and repeated A and C fibre stimulation remained unaffected. These results show that early skin injury can cause prolonged changes in central sensory connections that persist into adult life, long after the skin has healed. Enlarged dorsal horn neuron receptive field sizes provide a physiological mechanism for the persistent behavioural hypersensitivity that follows neonatal skin injury in rats and for the prolonged sensory changes reported in human infants after early pain and injury.
Store‐operated Ca2+ entry in first trimester and term human placenta
Authors: Clarson L. H. , Roberts V. H. J. , Hamark B. , Elliott A. C. , Powell T.
Published: May 2003

We have examined whether store‐operated Ca2+ entry, a common pathway for Ca2+ entry in non‐excitable tissue, is apparent in the syncytiotrophoblast of both first trimester and term human placenta. Expression of transient receptor potential (TRPC) homologues, a family of channels thought to be involved in store‐operated Ca2+ entry, was also studied at the mRNA and protein levels. [Ca2+]i in syncytiotrophoblast of first trimester and term placental villous fragments was measured by microfluorimetry using the Ca2+‐sensitive dye fura‐2. Store‐operated Ca2+ entry was stimulated using 1 μM thapsigargin in Ca2+‐free Tyrode buffer (no added Ca2++ 1 mM EGTA) followed by superfusion with control (Ca2+‐containing) buffer. In term fragments, this protocol resulted in a rapid increase in [Ca2+]i, which was inhibited in the presence of 150 μM GdCl3, 200 μM NiCl2, 200 μM CoCl2 or 30 μM SKF96365 but was unaffected by addition of 10 μM nifedipine. It was not possible to stimulate such a rise in [Ca2+]i in first trimester fragments. Messenger RNA encoding TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was identified in both first trimester and term placentas. From Western blotting, TRPC3 and TRPC6 proteins were detected in term, but not in first trimester, placentas, while TRPC1 protein was not detected. By immunocytochemistry, TRPC3 and TRPC4 were localised to cytotrophoblast cells in first trimester placentas and to the syncytiotrophoblast in term placentas. TRPC6 staining was present in the syncytiotrophoblast of both first trimester and term placenta, but the intensity was much greater in the latter. We propose that store‐operated Ca2+ entry may be an important route for Ca2+ entry into the syncytiotrophoblast of term, but not first trimester placentas, and that in human placenta TRPC channels may underlie this entry mechanism.
Transmission Dynamics and Control of Severe Acute Respiratory Syndrome
Authors: Lipsitch Marc , Cohen Ted , Cooper Ben , Robins James M. , Ma Stefan , James Lyn , Gopalakrishna Gowri , Chew Suok Kai , Tan Chorh Chuan , Samore Matthew H. , Fisman David , Murray Megan
Published: May 2003

Severe acute respiratory syndrome (SARS) is a recently described illness of humans that has spread widely over the past 6 months. With the use of detailed epidemiologic data from Singapore and epidemic curves from other settings, we estimated the reproductive number for SARS in the absence of interventions and in the presence of control efforts. We estimate that a single infectious case of SARS will infect about three secondary cases in a population that has not yet instituted control measures. Public-health efforts to reduce transmission are expected to have a substantial impact on reducing the size of the epidemic.
Inactivation of Mg Chelatase during Transition from Anaerobic to Aerobic Growth in
Rhodobacter capsulatus
Authors: Willows Robert D. , Lake Vanessa , Roberts Thomas Hugh , Beale Samuel I.
Published: May 2003

The facultative photosynthetic bacterium
Rhodobacter capsulatus
can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg
2+
to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the
bchI
,
bchD
, and
bchH
genes in
R
.
capsulatus
. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from
Escherichia coli
is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O
2
, and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing
R
.
capsulatus
cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O
2
-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.
Functional Subsets of the VirB Type IV Transport Complex Proteins Involved in the Capacity ofAgrobacterium tumefaciensTo Serve as a Recipient invirB-Mediated Conjugal Transfer of Plasmid RSF1010
Authors: Liu Zhenying , Binns Andrew N.
Published: May 2003

ThevirB-encoded type IV transport complex ofAgrobacterium tumefaciensmediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, betweenAgrobacteriumstrains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups ofvirBgenes revealed that translation ofvirB6is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient invirB-mediated conjugal RSF1010 transfer.
Phosphorylation of the Lipid A Region of Meningococcal Lipopolysaccharide: Identification of a Family of Transferases That Add Phosphoethanolamine to Lipopolysaccharide
Authors: Cox Andrew D. , Wright J. Claire , Li Jianjun , Hood Derek W. , Moxon E. Richard , Richards James C.
Published: May 2003

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase,
lpt-3
, has been identified from the
Neisseria meningitidis
genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and
lpt-3
represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions,
N. meningitidis
elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of “phosphoforms” that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene “
lptA
,” for “LPS phosphoethenolamine transferase for lipid A.”
Transcription of Bacteriophage PM2 Involves Phage-Encoded Regulators of Heterologous Origin
Authors: Männistö Riina H. , Grahn A. Marika , Bamford Dennis H. , Bamford Jaana K. H.
Published: May 2003

Bacteriophage PM2 is the only described member of the
Corticoviridae
family. It is an icosahedral dsDNA virus with a membrane residing underneath the protein coat. PM2 infects some gram-negative
Pseudoalteromonas
spp. In the present study, we mapped the viral promoters and showed that the PM2 genome consists of three operons. Four new virus genes were assigned based on their function in transcription. Proteins P15 and P16 are shown to repress early transcription, and proteins P13 and P14 are shown to activate late transcription events. The early regulatory region, containing genes for proteins P15 and P16, as well as the newly identified early promoter region in PM2, has significant sequence similarity with the
Pseudoalteromonas
pAS28 plasmid. P14, the transcription activator for the structural genes, has a zinc finger motif homologous to archaeal and eukaryotic TFIIS-type regulatory factors.
Contribution of Aggregation-Promoting Factor to Maintenance of Cell Shape in
Lactobacillus
gasseri
4B2
Authors: Jankovic Ivana , Ventura Marco , Meylan Valerie , Rouvet Martine , Elli Marina , Zink Ralf
Published: May 2003

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of
Lactobacillus
gasseri
4B2, whose corresponding
apf
gene was cloned and sequenced. In this report, we identified and sequenced an additional
apf
gene located in the region upstream of the previously published one. Inactivation of both
apf
genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the
apf2
promoter region on a high-copy-number plasmid, which recruited a putative
apf
activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for
L
.
gasseri
4B2 cell shape maintenance.
Impaired Growth Rates in Milk of
Lactobacillus helveticus
Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements
Authors: Christensen Jeffrey E. , Steele James L.
Published: May 2003

To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph
Lactobacillus helveticus
CNRZ32, single- and multiple-peptidase-deletion mutants were constructed. Two broad-specificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis. The inactivation of all three peptidases in JLS247 (Δ
pepC
Δ
pepN
Δ
pepX
) did not affect the growth rate in amino acid-defined medium. However, the peptidase mutants generally had decreased specific growth rates when acquisition of amino acids required hydrolysis of the proteins in milk, the most significant result being a 73% increase in generation time for JLS247. The growth rate deficiencies in milk were overcome by amino acid supplements with some specificity to each of the peptidase mutants. For example, milk supplementation with Pro resulted in the most significant growth rate increase for Δ
pepX
strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val) resulted in a JLS247 growth rate indistinguishable from that of the wild type. Our results show that characterization of the activities of the broad-specificity aminopeptidases had little predictive value regarding the amino acid supplements found to enhance the milk growth rates of the peptidase mutant strains. These results represent the first determination of the physiological roles with respect to specific amino acid requirements for peptidase mutants grown in milk.
How Clonal Is
Staphylococcus aureus
?
Authors: Feil Edward J. , Cooper Jessica E. , Grundmann Hajo , Robinson D. Ashley , Enright Mark C. , Berendt Tony , Peacock Sharon J. , Smith John Maynard , Murphy Michael , Spratt Brian G. , Moore Catrin E. , Day Nicholas P. J.
Published: May 2003

Staphylococcus aureus
is an important human pathogen and represents a growing public health burden owing to the emergence and spread of antibiotic-resistant clones, particularly within the hospital environment. Despite this, basic questions about the evolution and population biology of the species, particularly with regard to the extent and impact of homologous recombination, remain unanswered. We address these issues through an analysis of sequence data obtained from the characterization by multilocus sequence typing (MLST) of 334 isolates of
S
.
aureus
, recovered from a well-defined population, over a limited time span. We find no significant differences in the distribution of multilocus genotypes between strains isolated from carriers and those from patients with invasive disease; there is, therefore, no evidence from MLST data, which index variation within the stable “core” genome, for the existence of hypervirulent clones of this pathogen. Examination of the sequence changes at MLST loci during clonal diversification shows that point mutations give rise to new alleles at least 15-fold more frequently than does recombination. This contrasts with the naturally transformable species
Neisseria meningitidis
and
Streptococcus pneumoniae
, in which alleles change between 5- and 10-fold more frequently by recombination than by mutation. However, phylogenetic analysis suggests that homologous recombination does contribute toward the evolution of this species over the long term. Finally, we note a striking excess of nonsynonymous substitutions in comparisons between isolates belonging to the same clonal complex compared to isolates belonging to different clonal complexes, suggesting that the removal of deleterious mutations by purifying selection may be relatively slow.
Novel Developmental Genes,
fruCD
, of
Myxococcus xanthus
: Involvement of a Cell Division Protein in Multicellular Development
Authors: Akiyama Takuya , Inouye Sumiko , Komano Teruya
Published: May 2003

Myxococcus xanthus
is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, two novel developmental genes,
fruC
and
fruD
, of
M. xanthus
were identified and characterized. The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including
Bacillus subtilis
. Vegetative cells of the
fruD
mutant exhibited a filamentous phenotype. The
fruC
and
fruD
mutants displayed similar delayed-development phenotypes. The formation of tightly aggregated mounds by
fruC
and
fruD
mutants was slower than that by the wild-type strain. Spore formation by the
fruC
and
fruD
mutants initiated after 30 h poststarvation, whereas wild-type
M. xanthus
initiated spore formation after 18 h. The
fruCD
genes were constitutively expressed as an operon during vegetative growth and development. S1 mapping revealed that transcription initiation sites of the
fruCD
operon were located 114 (P1) and 55 bp (P2) upstream of the
fruC
initiation codon. Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development. The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.
Genomic Sequence of C
1
, the First Streptococcal Phage
Authors: Nelson Daniel , Schuch Raymond , Zhu Shiwei , Tscherne Donna M. , Fischetti Vincent A.
Published: May 2003

C
1
, a lytic bacteriophage infecting group C streptococci, is one of the earliest-isolated phages, and the method of bacterial classification known as phage typing was defined by using this bacteriophage. We present for the first time a detailed analysis of this phage by use of electron microscopy, protein profiling, and complete nucleotide sequencing. This virus belongs to the
Podoviridae
family of phages, all of which are characterized by short, noncontractile tails. The C
1
genome consists of a linear double-stranded DNA molecule of 16,687 nucleotides with 143-bp inverted terminal repeats. We have assigned functions to 9 of 20 putative open reading frames based on experimental substantiation or bioinformatic analysis. Their products include DNA polymerase, holin, lysin, major capsid, head-tail connector, neck appendage, and major tail proteins. Additionally, we found one intron belonging to the HNH endonuclease family interrupting the apparent lysin gene, suggesting a potential splicing event yielding a functional lytic enzyme. Examination of the C
1
DNA polymerase suggests that this phage utilizes a protein-primed mechanism of replication, which is prominent in the φ29-like members of
Podoviridae
. Consistent with this evidence, we experimentally determined that terminal proteins are covalently attached to both 5′ termini, despite the fact that no homology to known terminal proteins could be elucidated in any of our open reading frames. Likewise, comparative genomics revealed no close evolutionary matches, suggesting that the C
1
bacteriophage is a unique member of the
Podoviridae
.
Distribution and Organization of Auxotrophic Genes on the Multichromosomal Genome of
Burkholderia multivorans
ATCC 17616
Authors: Komatsu Harunobu , Imura Yoshiyuki , Ohori Akira , Nagata Yuji , Tsuda Masataka
Published: May 2003

The
Burkholderia multivorans
strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this β-proteobacterium, various auxotrophic mutations were isolated using a Tn
5
derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the
his
,
trp
,
arg
, and
lys
mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine
his
genes were clustered on the 3.4-Mb chromosome; (ii) seven
trp
genes were organized within two distinct regions, i.e., a
trpEGDC
cluster on the 3.4-Mb chromosome and a
trpFBA
cluster on the 2.5-Mb chromosome; (iii) the
leu
gene cluster,
leuCDB
, was also located close to the
trpFBA
cluster; and (iv)
lysA
and
argG
genes were located on the 2.5-Mb chromosome, in contrast to the
argH
gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.
Concentration and Assembly of the Division Ring Proteins FtsZ, FtsA, and ZipA during theEscherichia coliCell Cycle
Authors: Rueda Sonsoles , Vicente Miguel , Mingorance Jesús
Published: May 2003

The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during theEscherichia coliB/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.
Genomic Analysis and Initial Characterization of the Chitinolytic System of
Microbulbifer degradans
Strain 2-40
Authors: Howard Michael B. , Ekborg Nathan A. , Taylor Larry E. , Weiner Ronald M. , Hutcheson Steven W.
Published: May 2003

The marine bacterium
Microbulbifer degradans
strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in
N
-acetyl-
d
-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-β-
d
-
N
,
N
′-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-β-
d
-
N
,
N
′,
N
"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB)
N
-acetyl-β-
d
-glucosaminidases and one cytoplasmic (HexC)
N
-acetyl-β-
d
-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the
Escherichia coli
K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a
nagA
mutation in
E
.
coli
K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.
The Dithiol:Disulfide Oxidoreductases DsbA and DsbB of
Rhodobacter capsulatus
Are Not Directly Involved in Cytochrome
c
Biogenesis, but Their Inactivation Restores the Cytochrome
c
Biogenesis Defect of CcdA-Null Mutants
Authors: Deshmukh Meenal , Turkarslan Serdar , Astor Donniel , Valkova-Valchanova Maria , Daldal Fevzi
Published: May 2003

The cytoplasmic membrane protein CcdA and its homologues in other species, such as DsbD of
Escherichia coli
, are thought to supply the reducing equivalents required for the biogenesis of
c
-type cytochromes that occurs in the periplasm of gram-negative bacteria. CcdA
-
null mutants of the facultative phototroph
Rhodobacter capsulatus
are unable to grow under photosynthetic conditions (Ps
−
) and do not produce any active cytochrome
c
oxidase (Nadi
−
) due to a pleiotropic cytochrome
c
deficiency. However, under photosynthetic or respiratory growth conditions, these mutants revert frequently to yield Ps
+
Nadi
+
colonies that produce
c
-type cytochromes despite the absence of CcdA. Complementation of a CcdA-null mutant for the Ps
+
growth phenotype was attempted by using a genomic library constructed with chromosomal DNA from a revertant. No complementation was observed, but plasmids that rescued a CcdA-null mutant for photosynthetic growth by homologous recombination were recovered. Analysis of one such plasmid revealed that the rescue ability was mediated by open reading frame 3149, encoding the dithiol:disulfide oxidoreductase DsbA. DNA sequence data revealed that the
dsbA
allele on the rescuing plasmid contained a frameshift mutation expected to produce a truncated, nonfunctional DsbA. Indeed, a
dsbA ccdA
double mutant was shown to be Ps
+
Nadi
+
, establishing that in
R. capsulatus
the inactivation of
dsbA
suppresses the
c
-type cytochrome deficiency due to the absence of
ccdA
. Next, the ability of the wild-type
dsbA
allele to suppress the Ps
+
growth phenotype of the
dsbA ccdA
double mutant was exploited to isolate
dsbA
-independent
ccdA
revertants. Sequence analysis revealed that these revertants carried mutations in
dsbB
and that their Ps
+
phenotypes could be suppressed by the wild-type allele of
dsbB
. As with
dsbA
, a
dsbB ccdA
double mutant was also Ps
+
Nadi
+
and produced
c
-type cytochromes. Therefore, the absence of either DsbA or DsbB restores
c
-type cytochrome biogenesis in the absence of CcdA. Finally, it was also found that the DsbA-null and DsbB-null single mutants of
R. capsulatus
are Ps
+
and produce
c
-type cytochromes, unlike their
E. coli
counterparts, but are impaired for growth under respiratory conditions. This finding demonstrates that in
R. capsulatus
the dithiol:disulfide oxidoreductases DsbA and DsbB are not essential for cytochrome
c
biogenesis even though they are important for respiration under certain conditions.
Genes of
Bacillus cereus
and
Bacillus anthracis
Encoding Proteins of the Exosporium
Authors: Todd Sarah J. , Moir Arthur J. G. , Johnson Matt J. , Moir Anne
Published: May 2003

The exosporium is the outermost layer of spores of
Bacillus cereus
and its close relatives
Bacillus anthracis
and
Bacillus thuringiensis
. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of
B. cereus
ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of
B. anthracis
or
B. cereus
ATCC 14579, and the precise corresponding sequence from
B. cereus
ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (
exsB
,
exsC
,
exsD
,
exsE
,
exsF
,
exsG
,
exsJ
, and
cotE
). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of
B. subtilis
, but most do not have homologues in
B. subtilis
. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of
B. anthracis
. Although most of the exosporium genes are scattered on the genome,
bclA
and
exsF
are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.
Sigma 54 Levels and Physiological Control of the
Pseudomonas putida
Pu Promoter
Authors: Jurado Paola , Fernández Luis A. , de Lorenzo Víctor
Published: May 2003

The cellular levels of the alternative sigma factor σ
54
of
Pseudomonas putida
have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of σ
54
were also monitored in
P. putida
strains with knockout mutations in
ptsO
or
ptsN
, known to be required for the C-source control of the σ
54
-dependent Pu promoter of the TOL plasmid. Our results show that ∼80 ± 26 molecules of σ
54
exist per cell. Unlike that in relatives of
Pseudomonas
(e.g.,
Caulobacter
), where fluctuations of σ
54
determine adaptation and differentiation when cells face starvation, σ
54
in
P. putida
remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the
ptsO
and
ptsN
mutant strains analyzed. The number of σ
54
molecules per cell in
P. putida
is barely above the predicted number of σ
54
-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other σ
54
-dependent systems) may become amenable to physiological control.
Polar Localization of Replicon Origins in the Multipartite Genomes ofAgrobacterium tumefaciensandSinorhizobium meliloti
Authors: Kahng Lyn Sue , Shapiro Lucy
Published: May 2003

The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated. In particular, little is known about the replication of multipartite genomes in bacteria. We determined the cellular positions of the origins of the replicons in the alpha proteobacteriaAgrobacterium tumefaciensandSinorhizobium melilotiand found that they are located at the poles of the cells. Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from therepABCfamily. The cellular location of a derivative of the RK2 plasmid is distinct from that of the alpha proteobacterium genomic replicon origins but is conserved across bacteria. Colocalization experiments with the genomic replicons ofA. tumefaciensrevealed that therepABCreplicons, although preferentially positioned at the cell pole, colocalize only rarely. For therepABCreplicons in this organism, occupying discrete spatial locations may contribute to their coexistence and stable inheritance.
Modeling Bacterial Evolution with Comparative-Genome-Based Marker Systems: Application to
Mycobacterium tuberculosis
Evolution and Pathogenesis
Authors: Alland David , Whittam Thomas S. , Murray Megan B. , Cave M. Donald , Hazbon Manzour H. , Dix Kim , Kokoris Mark , Duesterhoeft Andreas , Eisen Jonathan A. , Fraser Claire M. , Fleischmann Robert D.
Published: May 2003

The comparative-genomic sequencing of two
Mycobacterium tuberculosis
strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical
M
.
tuberculosis
. Phylogenetic analysis using these “comparative-genome markers” (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches. To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format. We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared “reference strains.” Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse. Improperly distanced reference strains generate CGMs that distort and reroot outgroups. Applying this understanding to the CGM-based phylogeny of
M
.
tuberculosis
, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange. We noted indications of evolutionary bottlenecks, including one at the level of the PHRI “C” strain previously associated with particular virulence characteristics. Our evidence also suggests that loss of IS
6110
to fewer than seven elements per genome is uncommon. Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure.
Coaggregation-Mediated Interactions of Streptococci and Actinomyces Detected in Initial Human Dental Plaque
Authors: Palmer, Robert J. , Gordon Sharon M. , Cisar John O. , Kolenbrander Paul E.
Published: May 2003

Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria. These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque. To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips. In intrageneric coaggregation, streptococci such as
Streptococcus gordonii
DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as
Streptococcus oralis
34. To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against
S. gordonii
DL1 (anti-DL1). These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque. Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains. In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci. Cells reactive with antibody against type 2 fimbriae of
Actinomyces naeslundii
T14V (anti-type-2) were much less frequent than either subset of streptococci. However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells. These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo. Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.
Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in
Escherichia coli
Authors: Rock Charles O. , Park Hee-Won , Jackowski Suzanne
Published: May 2003

Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in
Escherichia coli
, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [
coaA15
(Ts)
panD2
]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4′-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.
Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides
Authors: Vignon Guillaume , Köhler Rolf , Larquet Eric , Giroux Stéphanie , Prévost Marie-Christine , Roux Pascal , Pugsley Anthony P.
Published: May 2003

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown
Escherichia
coli
K-12 cells expressing the
Klebsiella
oxytoca
pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in
E
.
coli
or to PulG. For example, high-level expression of the endogenous
E
.
coli
gsp
secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic “pseudopilus” and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).
Cysteine-Scanning Analysis of the Dimerization Domain of EnvZ, an Osmosensing Histidine Kinase
Authors: Qin Ling , Cai Shengjian , Zhu Yan , Inouye Masayori
Published: May 2003

EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in
Escherichia coli
. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.
Open Reading Frame
sso2387
from the Archaeon
Sulfolobus solfataricus
Encodes a Polypeptide with Protein-Serine Kinase Activity
Authors: Lower Brian H. , Kennelly Peter J.
Published: May 2003

The predicted polypeptide product of open reading frame
sso2387
from the archaeon
Sulfolobus solfataricus
, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily.
sso2387
was cloned, and its polypeptide product was expressed in
Escherichia coli
. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser
548
, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.
Quantitation of
mecA
Transcription in Oxacillin-Resistant
Staphylococcus aureus
Clinical Isolates
Authors: Rosato Adriana E. , Craig William A. , Archer Gordon L.
Published: May 2003

The transcription of
mecA
, the gene required for oxacillin resistance in staphylococci, was quantified in a collection of 65 geographically and genetically diverse clinical and 8 defined laboratory
Staphylococcus aureus
isolates.
mecA
transcription was measured by real-time reverse transcription-PCR, confirmed by Northern blot analysis, and correlated with the presence and DNA sequence of the two
mecA
repressors,
mecI
and
blaI
. Isolates were first examined that contained
mecI
and/or
blaI
with wild-type sequence. BlaI provided significantly more repression of
mecA
transcription than did MecI, unrelated to
blaI
genetic location. Both together repressed
mecA
better than either one alone. In clinical isolates containing only wild-type
mecI
,
mecA
transcription repression was 10- to 25-fold less effective than that seen in previously studied constructs derived from strain N315. There was a difference in the
mecI
ribosomal binding site (RBS) between the clinical isolates (GGAA) and N315 (GGAG). The GGAA RBS was associated with 5.5- to 7.3-fold less
mecA
repression than GGAG in isogenic constructs. The values generated for wild-type repressors were compared to those in 26 isolates containing
mecI
mutations.
mecA
transcription appeared to be repressed only by BlaI in isolates with
mecI
nonsense and frameshift mutations. In contrast,
mecI
repression seemed to be partially or fully retained in many of the isolates with
mecI
and one isolate with
blaI
missense mutations, providing structure-function correlates with the site and type of mutation. We conclude that
mecA
repressor activity is highly variable in clinical
S. aureus
isolates due to
mecI
mutations, RBS polymorphisms, and unidentified genomic adaptations.
Conserved Eukaryotic Histone-Fold Residues Substituted into an Archaeal Histone Increase DNA Affinity but Reduce Complex Flexibility
Authors: Soares Divya J. , Marc Frédéric , Reeve John N.
Published: May 2003

Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.
Tertiary Structure of Bacterial Murein: the Scaffold Model
Authors: Dmitriev Boris A. , Toukach Filip V. , Schaper Klaus-Jürgen , Holst Otto , Rietschel Ernst T. , Ehlers Stefan
Published: May 2003

Although the chemical structure and physical properties of peptidoglycan have been elucidated for some time, the precise three-dimensional organization of murein has remained elusive. Earlier published computer simulations of the bacterial murein architecture modeled peptidoglycan strands in either a regular (D. Pink, J. Moeller, B. Quinn, M. Jericho, and T. Beveridge, J. Bacteriol. 182: 5925-5930, 2000) or an irregular (A. Koch, J. Theor. Biol. 204: 533-541, 2000) parallel orientation with respect to the plasma membrane. However, after integrating published experimental data on glycan chain length distribution and the degree of peptide side chain cross-linking into this computer simulation, we now report that the proposed planar network of murein appears largely dysfunctional. In contrast, a scaffold model of murein architecture, which assumes that glycan strands extend perpendicularly to the plasma membrane, was found to accommodate published experimental evidence and yield a viable stress-bearing matrix. Moreover, this model is in accordance with the well-established principle of murein assembly in vivo, i.e., sequential attachment of strands to the preexisting structure. For the first time, the phenomenon of division plane alternation in dividing bacteria can be reconciled with a computer model of the molecular architecture of murein.