bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of an Efficient
In Vivo
System (P
junc
-TpaseIS

1223

) for Random Transposon Mutagenesis of Lactobacillus casei
Authors: Licandro-Seraut Hélène , Brinster Sophie , van de Guchte Maarten , Scornec Hélène , Maguin Emmanuelle , Sansonetti Philippe , Cavin Jean-François , Serror Pascale 
Published: May 2012

ABSTRACT

The random transposon mutagenesis system P
junc
-TpaseIS

1223

is composed of plasmids pVI129, expressing IS
1223
transposase, and pVI110, a suicide transposon plasmid carrying the P
junc
sequence, the substrate of the IS
1223
transposase. This system is particularly efficient in
Lactobacillus casei
, as more than 10,000 stable, random mutants were routinely obtained via electroporation.
Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of
l
-Lysine and
l
-Glutamate from Methanol
Authors: Heggeset Tonje M. B. , Krog Anne , Balzer Simone , Wentzel Alexander , Ellingsen Trond E. , Brautaset Trygve 
Published: May 2012

ABSTRACT

Bacillus methanolicus
can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into
l
-lysine and
l
-glutamate. We present here the genome sequences of the important
B. methanolicus
model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their
l
-glutamate production levels (406 mmol liter
−1
and 11 mmol liter
−1
, respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for
l
-lysine and
l
-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two
B. methanolicus
strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of
B. methanolicus
and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.
Comparative Genomic Analyses of 17 Clinical Isolates of Gardnerella vaginalis Provide Evidence of Multiple Genetically Isolated Clades Consistent with Subspeciation into Genovars
Authors: Ahmed Azad , Earl Josh , Retchless Adam , Hillier Sharon L. , Rabe Lorna K. , Cherpes Thomas L. , Powell Evan , Janto Benjamin , Eutsey Rory , Hiller N. Luisa , Boissy Robert , Dahlgren Margaret E. , Hall Barry G. , Costerton J. William , Post J. Christopher , Hu Fen Z. , Ehrlich Garth D. 
Published: May 2012

ABSTRACT

Gardnerella vaginalis
is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen
G. vaginalis
isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the
G. vaginalis
strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets.
G. vaginalis
appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.
CcpA-Dependent Carbohydrate Catabolite Repression Regulates Galactose Metabolism in Streptococcus oligofermentans
Authors: Cai Jun , Tong Huichun , Qi Fengxia , Dong Xiuzhu 
Published: May 2012

ABSTRACT

Streptococcus oligofermentans
is an oral commensal that inhibits the growth of the caries pathogen
Streptococcus mutans
by producing copious amounts of H
2
O
2
and that grows faster than
S. mutans
on galactose. In this study, we identified a novel eight-gene galactose (
gal
) operon in
S. oligofermentans
that was comprised of
lacABCD
,
lacX
, and three genes encoding a galactose-specific transporter. Disruption of
lacA
caused more growth reduction on galactose than mutation of
galK
, a gene in the Leloir pathway, indicating that the principal role of this operon is in galactose metabolism. Diauxic growth was observed in cultures containing glucose and galactose, and a luciferase reporter fusion to the putative
gal
promoter demonstrated 12-fold repression of the operon expression by glucose but was induced by galactose, suggesting a carbon catabolite repression (CCR) control in galactose utilization. Interestingly, none of the single-gene mutations in the well-known CCR regulators
ccpA
and
manL
affected diauxic growth, although the operon expression was upregulated in these mutants in glucose. A double mutation of
ccpA
and
manL
eliminated glucose repression of galactose utilization, suggesting that these genes have parallel functions in regulating
gal
operon expression and mediating CCR. Electrophoretic mobility shift assays demonstrated binding of CcpA to the putative catabolite response element motif in the promoter regions of the
gal
operon and
manL
, suggesting that CcpA regulates CCR through direct regulation of the transcription of the
gal
operon and
manL
. This provides the first example of oral streptococci using two parallel CcpA-dependent CCR pathways in controlling carbohydrate metabolism.
Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition
Authors: Zoll Sebastian , Schlag Martin , Shkumatov Alexander V. , Rautenberg Maren , Svergun Dmitri I. , Götz Friedrich , Stehle Thilo 
Published: May 2012

ABSTRACTThe bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division.
Development of Competence for Genetic Transformation of
Streptococcus mutans
in a Chemically Defined Medium
Authors: Desai Kunal , Mashburn-Warren Lauren , Federle Michael J. , Morrison Donald A. 
Published: May 2012

ABSTRACT

Streptococcus mutan
s develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun.
27:
444, 1980). We confirmed a previous report that in this medium
comS
mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS
11-17
) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that
comS
+
(but not
comS
mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a
comS
mutation at levels equivalent to 1 μM synthetic ComS
11-17
.
DprB Facilitates Inter- and Intragenomic Recombination in Helicobacter pylori
Authors: Zhang Xue-Song , Blaser Martin J. 
Published: May 2012

ABSTRACT

For naturally competent microorganisms, such as
Helicobacter pylori
, the steps that permit recombination of exogenous DNA are not fully understood. Immediately downstream of an
H. pylori
gene (
dprA
) that facilitates high-frequency natural transformation is HP0334 (
dprB
), annotated to be a putative Holliday junction resolvase (HJR). We showed that the HP0334 (
dprB
) gene product facilitates high-frequency natural transformation. We determined the physiologic roles of DprB by genetic analyses. DprB controls
in vitro
growth, survival after exposure to UV or fluoroquinolones, and intragenomic recombination.
dprB ruvC
double deletion dramatically decreases both homologous and homeologous transformation and survival after exposure to DNA-damaging agents. Moreover, the DprB protein binds to synthetic Holliday junction structures rather than double-stranded or single-stranded DNA. These results demonstrate that the
dprB
product plays important roles affecting inter- and intragenomic recombination. We provide evidence that the two putative
H. pylori
HJRs (DprB and RuvC) have overlapping but distinct functions involving intergenomic (primarily DprB) and intragenomic (primarily RuvC) recombination.
Secretion Genes as Determinants of Bacillus anthracis Chain Length
Authors: Nguyen-Mau Sao-Mai , Oh So-Young , Kern Valerie J. , Missiakas Dominique M. , Schneewind Olaf 
Published: May 2012

ABSTRACT

Bacillus anthracis
grows in chains of rod-shaped cells, a trait that contributes to its escape from phagocytic clearance in host tissues. Using a genetic approach to search for determinants of
B. anthracis
chain length, we identified mutants with insertional lesions in
secA2
. All isolated
secA2
mutants exhibited an exaggerated chain length, whereas the dimensions of individual cells were not changed. Complementation studies revealed that
slaP
(
S
-
l
ayer
a
ssembly
p
rotein), a gene immediately downstream of
secA2
on the
B. anthracis
chromosome, is also a determinant of chain length. Both
secA2
and
slaP
are required for the efficient secretion of Sap and EA1 (Eag), the two S-layer proteins of
B. anthracis
, but not for the secretion of S-layer-associated proteins or of other secreted products. S-layer assembly via
secA2
and
slaP
contributes to the proper positioning of BslO, the S-layer-associated protein, and murein hydrolase, which cleaves septal peptidoglycan to separate chains of bacilli. SlaP was found to be both soluble in the bacterial cytoplasm and associated with the membrane. The purification of soluble SlaP from
B. anthracis
-cleared lysates did not reveal a specific ligand, and the membrane association of SlaP was not dependent on SecA2, Sap, or EA1. We propose that SecA2 and SlaP promote the efficient secretion of S-layer proteins by modifying the general secretory pathway of
B. anthracis
to transport large amounts of Sap and EA1.
Degradation of 2,3-Dihydroxybenzoate by a Novel
meta
-Cleavage Pathway
Authors: Marín Macarena , Plumeier Iris , Pieper Dietmar H. 
Published: May 2012

ABSTRACT

2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via
meta
-cleavage of the aromatic ring. The
dhb
cluster of
Pseudomonas reinekei
MT1 encodes a chimeric
meta
-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-
p
-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of
P. reinekei
MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the
dhB
gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-
p
-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate.
Two Surfaces of a Conserved Interdomain Linker Differentially Affect Output from the RST Sensing Module of the Bacillus subtilis Stressosome
Authors: Gaidenko Tatiana A. , Bie Xiaomei , Baldwin Enoch P. , Price Chester W. 
Published: May 2012

ABSTRACT

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ
B
stress factor of
Bacillus subtilis
. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the
in vivo
role of the linker using alanine scanning mutagenesis, assaying stressosome output in
B. subtilis
via a σ
B
-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer
in vivo
, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.
Selenium-Dependent Biogenesis of Formate Dehydrogenase in
Campylobacter jejuni
Is Controlled by the
fdhTU
Accessory Genes
Authors: Shaw Frances L. , Mulholland Francis , Le Gall Gwénaëlle , Porcelli Ida , Hart Dave J. , Pearson Bruce M. , van Vliet Arnoud H. M. 
Published: May 2012

ABSTRACT

The food-borne bacterial pathogen
Campylobacter jejuni
efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the
cj1500
and
cj1501
genes of
C. jejuni
, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names
fdhT
and
fdhU
, respectively. Insertional inactivation of
fdhT
or
fdhU
in
C. jejuni
resulted in the absence of FdhA and FdhB protein expression, reduced
fdhABC
RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by
1
H nuclear magnetic resonance. The
fdhABC
genes are transcribed from a single promoter located two genes upstream of
fdhA
, and the decrease in
fdhABC
RNA levels in the
fdhU
mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with
fdhU
and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the
selA
and
selB
genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of
selA
and
fdhTU
orthologs and the predicted presence of SeC in FdhA. The
fdhTU
genes encode accessory proteins required for FDH expression and activity in
C. jejuni
, possibly by contributing to acquisition or utilization of selenium.
Consent to tissue banking for research: qualitative study and recommendations
Authors: Soto Carmen , Tarrant Carolyn , Pritchard-Jones Kathy , Dixon-Woods Mary 
Published: May 2012

ObjectiveTo explore how families of children with cancer experience giving consent for tissue banking and to produce recommendations on good practice.Design79 participants from 42 families (41 mothers, 18 fathers, 20 children and young people with cancer) took part in semistructured interviews to explore their experiences of being approached for consent to tissue banking.SettingTertiary care facilities for childhood cancer.ResultsFamilies are generally supportive of tissue banking, although they report that it may be difficult for them to consider all the implications when asked for consent. They typically do not want detailed information when consent is sought close to diagnosis, preferring to see tissue banking as part of routine practice. Families often recognise that their consent may not be fully informed, but are content to give consent based on their understanding at the time. Some may want a chance to go over the information and revisit their decision when things have settled.ConclusionsFamilies' views can inform practical recommendations for optimising the experience of consent for tissue banking. Current guidelines for obtaining consent should be revisited to take account of families' preferences.
Managing patient deterioration: assessing teamwork and individual performance
Authors: Cooper Simon , Cant Robyn , Porter Jo , Missen Karen , Sparkes Louise , McConnell-Henry Tracy , Endacott Ruth 
Published: May 2012

ObjectiveTo assess the ability of rural Australian nurse teams to manage deteriorating patients.MethodsThis quasi-experimental design used pre- and post-intervention assessments and observation to evaluate nurses' simulated clinical performance. Registered nurses (n=44) from two hospital wards completed a formative knowledge assessment and three team-based video recorded scenarios (Objective Structured Clinical Examinations (OSCE)). Trained patient actors simulated deteriorating patients. Skill performance and situation awareness were measured and team performance was rated using the Team Emergency Assessment Measure.ResultsKnowledge in relation to patient deterioration management varied (mean 63%, range 27–100%) with a median score of 64%. Younger nurses with a greater number of working hours scored the highest (p=0.001). OSCE performance was generally low with a mean performance of 54%, but performance was maintained despite the increasing complexity of the scenarios. Situation awareness was generally low (median 50%, mean 47%, range 17–83%, SD 14.03) with significantly higher levels in younger participants (r=−0.346, p=0.021). Teamwork ratings averaged 57% with significant associations between the subscales (Leadership, Teamwork and Task Management) (p<0.006), the global rating scale (p<0.001) and two of the OSCE measures (p<0.049). Feedback from participants following the programme indicated significant improvements in knowledge, confidence and competence (p<0.001).ConclusionDespite a satisfactory knowledge base, the application of knowledge was low with notable performance deficits in these demanding and stressful situations. The identification and management of patient deterioration needs to be taught in professional development programmes incorporating high fidelity simulation techniques. The Team Emergency assessment tool proved to be a valid measure of team performance in patient deterioration scenarios.
Implementation of a patient selection protocol for intra-arterial therapy increases treatment rates in patients with acute ischemic stroke
Authors: Rost Natalia S , Smith Eric E , Nogueira Raul G , Fitzpatrick Kaitlin M , Yoo Albert J , Hirsch Joshua A , Schwamm Lee H 
Published: May 2012

BackgroundStrategies for patient selection for intra-arterial therapy (IAT) in acute ischemic stroke (AIS) are highly variable. The degree of protocol adoption and treatment rates associated with implementation of a service-wide patient selection IAT protocol were assessed.MethodsAll patients with AIS prospectively recorded in our stroke database from January 2007 to June 2009 were reviewed. The IAT patient selection protocol was implemented in March 2008. Patients were defined as likely to benefit (LTB) from IAT if they had brain imaging completed within 6 h from last known well time, NIH Stroke Scale score ≥8, infarct volume ≤100 ml and evidence of proximal artery occlusion.ResultsOf 1348 subjects identified, 118 (8.7%) met the criteria for LTB and 62 (52%) underwent IAT. There was a significant increase in rates of IAT among LTB patients after protocol implementation (61% vs 40%, p<0.02). In LTB patients, factors associated with IAT were stroke duration (OR 0.78, 95% CI 0.6 to 0.9 per hour), arrival within later calendar months during study period (OR 1.1, 95% CI 1.02 to 1.2 per month), intravenous tissue plasminogen activator (OR 0.6, 95% CI 0.4 to 0.9) and age (OR 0.98, 95% CI 0.95 to 1.02 per year). After multivariable adjustment, only stroke duration (OR 0.65, 95% CI 0.5 to 0.8 per hour) remained an independent predictor of IAT.ConclusionsMost patients with AIS did not meet our criteria for LTB and only 52% of those defined as LTB received IAT. Protocol adoption increased the use of IAT over time; however, further exploration of factors associated with the reasons for non-treatment and the impact of IAT on outcomes is necessary.