bluuebunny/crossref_metadata_2025_split huggingface dataset:
Listeria monocytogenes σHContributes to Expression of Competence Genes and Intracellular Growth
Authors: Medrano Romero Veronica , Morikawa Kazuya 
Published: February 2016

ABSTRACTThe alternative sigma factor σHhas two functions in Gram-positive bacteria: it regulates sporulation and the development of genetic competence.Listeria monocytogenesis a nonsporulating species in which competence has not yet been detected. Nevertheless, the main competence regulators and a series of orthologous genes that form the competence machinery are present in its genome; some of the competence genes play a role in optimal phagosomal escape. In this study, strains overexpressing σHand strains with a σHdeletion were used to elucidate the contribution of σHto the expression of the competence machinery genes inL. monocytogenes. Gene expression analysis showed that σHis, indeed, involved incomGandcomEregulation. Unexpectedly, we observed a unique regulation scheme in which σHand the transcription factor ComK were involved. Population-level analysis showed that even with the overexpression of both factors, only a fraction of the cells expressed the competence machinery genes. Although we could not detect competence, σHwas crucial for phagosomal escape, which implies that this alternative sigma factor has specifically evolved to regulate theL. monocytogenesintracellular life cycle.IMPORTANCEListeria monocytogenescan be an intracellular pathogen capable of causing serious infections in humans and animal species. Recently, the competence machinery genes were described as being necessary for optimal phagosomal escape, in which the transcription factor ComK plays an important role. On the other hand, our previous phylogenetic analysis suggested that the alternative sigma factor σHmight play a role in the regulation of competence genes. The present study shows that some of the competence genes belong to the σHregulon and, importantly, that σHis essential for intracellular growth, implying a unique physiological role of σHamongFirmicutes.
Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires
citT
and
dctA
Authors: Van Hofwegen Dustin J. , Hovde Carolyn J. , Minnich Scott A. 
Published: February 2016

ABSTRACT

The isolation of aerobic citrate-utilizing
Escherichia coli
(Cit
+
) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of
E. coli
Cit
+
mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type
E. coli
strain B and with K-12 and three K-12 derivatives: an
E. coli
Δ
rpoS
::
kan
mutant (impaired for stationary-phase survival), an
E. coli
Δ
citT
::
kan
mutant (deleted for the anaerobic citrate/succinate antiporter), and an
E. coli
Δ
dctA
::
kan
mutant (deleted for the aerobic succinate transporter).
E. coli
underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent
E. coli
Cit
+
mutants were isolated from all
E. coli
derivatives except the
E. coli
Δ
citT
::
kan
mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry.
E. coli
Cit
+
mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four
E. coli
Cit
+
mutants revealed the required sequence of mutational events leading to a refined Cit
+
mutant. These events showed amplified
citT
and
dctA
loci followed by DNA rearrangements consistent with promoter capture events for
citT
. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.

IMPORTANCE
E. coli
cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513–518, 2012,
http://dx.doi.org/10.1038/nature11514
) found a single aerobic, citrate-utilizing
E. coli
strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the
citT
and
dctA
loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.
The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA
Authors: Kuchinski Kevin S. , Brimacombe Cedric A. , Westbye Alexander B. , Ding Hao , Beatty J. Thomas 
Published: February 2016

ABSTRACT

The gene transfer agent of
Rhodobacter capsulatus
(RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding
R. capsulatus
cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the
lexA
gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the
R. capsulatus
genome sequence identified a number of putative binding sites located 5′ of typical SOS response coding sequences and also 5′ of the RcGTA regulatory gene
cckA
, which encodes a hybrid histidine kinase homolog. Expression of
cckA
was increased >5-fold in the
lexA
mutant, and a
lexA cckA
double mutant was found to have the same phenotype as a Δ
cckA
single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the
lexA
mutant is largely due to the overexpression of
cckA
.

IMPORTANCE
This work describes an unusual phenotype of a
lexA
mutant of the alphaproteobacterium
Rhodobacter capsulatus
in respect to the phage transduction-like genetic exchange carried out by the
R. capsulatus
gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this
lexA
mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically.
Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction
Authors: Baker Anna W. , Satyshur Kenneth A. , Moreno Morales Neydis , Forest Katrina T. 
Published: February 2016

ABSTRACT

Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium
Ramlibacter tataouinensis
. RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the
R. tataouinensis
bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from
Agrobacterium tumefaciens
, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRR
mon
) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems.

IMPORTANCE
BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.
Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex
Authors: Stevenson Begoña , Wyckoff Elizabeth E. , Payne Shelley M. 
Published: February 2016

ABSTRACT

Feo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. The
feo
operon in
Vibrio cholerae
is made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linked
in vivo
in
V. cholerae
. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport.

IMPORTANCE
The Feo system is the major route for ferrous iron transport in bacteria. In this work, the
Vibrio cholerae
Feo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complex
in vivo
. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly.
Colanic Acid Intermediates Prevent
De Novo
Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid
Authors: Ranjit Dev K. , Young Kevin D. 
Published: February 2016

ABSTRACT

After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In
Escherichia coli
, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse. Here, we investigated the reason for this Rcs requirement. Rcs-deficient spheroids exhibited breaks and bulges in their periplasmic spaces and failed to synthesize a complete peptidoglycan cell wall, indicating that the bacterial envelope was defective. To determine the Rcs-dependent gene(s) required for shape recovery, we tested spheroplasts lacking selected RcsB-regulated genes and found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, extracellular CA was not required for recovery. Instead, lysis was caused by mutations that interrupted CA biosynthesis downstream of the initial glycosyl transferase, WcaJ. Deleting
wcaJ
prevented lysis of spheroplasts lacking ensuing steps in the pathway, and providing WcaJ in
trans
to a mutant lacking the entire CA operon triggered spheroplast enlargement and lysis. Thus, CA is not required for spheroplast recovery. Instead, CA intermediates accumulate as dead-end products which inhibit recovery of wall-less cells. The results strongly imply that CA may not be required for the survival
E. coli
L-forms. More broadly, these findings mandate that previous conclusions about the role of colanic acid in biofilm formation or virulence must be reevaluated.

IMPORTANCE
Wall-less bacteria can resynthesize their walls and recreate a normal shape, which in
Escherichia coli
requires the Rcs response. While attempting to identify the Rcs-dependent gene required for shape recovery, we found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, cell death was caused by mutations that interrupted CA biosynthesis downstream of the initial step in the pathway, creating dead-end compounds that inhibited recovery of wall-less cells. When testing for the biological role of CA, most previous experiments used mutants that would accumulate these deadly intermediates, meaning that all prior conclusions must be reexamined to determine if the results were caused by these lethal side effects instead of accurately reflecting the biological purpose of CA itself.
The Composition of the Cell Envelope Affects Conjugation in Bacillus subtilis
Authors: Johnson Christopher M. , Grossman Alan D. 
Published: February 2016

ABSTRACTConjugation in bacteria is the contact-dependent transfer of DNA from one cell to another via donor-encoded conjugation machinery. It is a major type of horizontal gene transfer between bacteria. Conjugation of the integrative and conjugative element ICEBs1intoBacillus subtilisis affected by the composition of phospholipids in the cell membranes of the donor and recipient. We found that reduction (or elimination) of lysyl-phosphatidylglycerol caused by loss ofmprFcaused a decrease in conjugation efficiency. Conversely, alterations that caused an increase in lysyl-phosphatidylglycerol, including loss ofugtPor overproduction ofmprF, caused an increase in conjugation efficiency. In addition, we found that mutations that alter production of other phospholipids, e.g., loss ofclsAandyfnI, also affected conjugation, apparently without substantively altering levels of lysyl-phosphatidylglycerol, indicating that there are multiple pathways by which changes to the cell envelope affect conjugation. We found that the contribution ofmprFto conjugation was affected by the chemical environment. Wild-type cells were generally more responsive to addition of anions that enhanced conjugation, whereasmprFmutant cells were more sensitive to combinations of anions that inhibited conjugation at pH 7. Our results indicate thatmprFand lysyl-phosphatidylglycerol allow cells to maintain relatively consistent conjugation efficiencies under a variety of ionic conditions.IMPORTANCEHorizontal gene transfer is a driving force in microbial evolution, enabling cells that receive DNA to acquire new genes and phenotypes. Conjugation, the contact-dependent transfer of DNA from a donor to a recipient by a donor-encoded secretion machine, is a prevalent type of horizontal gene transfer. Although critically important, it is not well understood how the recipient influences the success of conjugation. We found that the composition of phospholipids in the membranes of donors and recipients influences the success of transfer of the integrative and conjugative element ICEBs1inBacillus subtilis. Specifically, the presence of lysyl-phosphatidylglycerol enables relatively constant conjugation efficiencies in a range of diverse chemical environments.
Are there three main subgroups within the patellofemoral pain population? A detailed characterisation study of 127 patients to help develop targeted intervention (TIPPs)
Authors: Selfe James , Janssen Jessie , Callaghan Michael , Witvrouw Erik , Sutton Chris , Richards Jim , Stokes Maria , Martin Denis , Dixon John , Hogarth Russell , Baltzopoulos Vasilios , Ritchie Elizabeth , Arden Nigel , Dey Paola 
Published: February 2016

BackgroundCurrent multimodal approaches for the management of non-specific patellofemoral pain are not optimal, however, targeted intervention for subgroups could improve patient outcomes. This study explores whether subgrouping of non-specific patellofemoral pain patients, using a series of low cost simple clinical tests, is possible.MethodThe exclusivity and clinical importance of potential subgroups was assessed by applying à priori test thresholds (1 SD) from seven clinical tests in a sample of adult patients with non-specific patellofemoral pain. Hierarchical clustering and latent profile analysis, were used to gain additional insights into subgroups using data from the same clinical tests.Results130 participants were recruited, 127 had complete data: 84 (66%) female, mean age 26 years (SD 5.7) and mean body mass index 25.4 (SD 5.83), median (IQR) time between onset of pain and assessment was 24 (7–60) months. Potential subgroups defined by the à priori test thresholds were not mutually exclusive and patients frequently fell into multiple subgroups. Using hierarchical clustering and latent profile analysis three subgroups were identified using 6 of the 7 clinical tests. These subgroups were given the following nomenclature: (1) ‘strong’, (2) ‘weak and tighter’ and (3) ‘weak and pronated foot’.ConclusionsWe conclude that three subgroups of patellofemoral patients may exist based on the results of six clinical tests which are feasible to perform in routine clinical practice. Further research is needed to validate these findings in other data sets and, if supported by external validation, to see if targeted interventions for these subgroups improve patient outcomes.
Return to work after knee replacement: a qualitative study of patient experiences
Authors: Bardgett Michelle , Lally Joanne , Malviya Ajay , Deehan David 
Published: February 2016

ObjectiveAn increasing number of patients in the working population are undergoing total knee replacement (TKR) for end-stage osteoarthritis. The timing and success of return to work is becoming increasingly important for this group of patients with social and economic implications for patients, employers and society. There is limited understanding of the patient variables that determine the ability to return to work. Our objective was (from the patient's perspective) to gain an insight into the factors influencing return to work following knee replacement.Setting and participantsThis qualitative study was undertaken in a secondary-care setting in a large teaching hospital in the north of England. Semistructured interviews were carried out with 10 patients regarding their experiences of returning to work following TKR.OutcomesInterviews were transcribed and analysed using a qualitative thematic approach to identify the factors influencing return to work from the patient's perspective.ResultsThree themes were identified that influenced the process of return to work, from the patient's perspective. These were delays in surgical intervention, limited and often inconsistent advice from healthcare professionals regarding return to work, and finally the absence of rehabilitation to optimise patient's recovery and facilitate return to work.ConclusionsThere is currently no consistent process to optimise return to work for patients of working age after TKR. The impact of delayed surgical intervention, limited advice regarding return to work, and a lack of work-focused rehabilitation, all contribute to potential delays in successful return to work. There is a need to change the focus of healthcare provision for this cohort of patients, and provide a tailored healthcare intervention to optimise patient outcomes.
A multinational cross-sectional survey of the management of patient medication adherence by European healthcare professionals
Authors: Clyne Wendy , Mshelia Comfort , McLachlan Sarah , Jones Peter , de Geest Sabina , Ruppar Todd , Siebens Kaat , Dobbels Fabienne , Kardas Przemyslaw 
Published: February 2016

ObjectivesTo examine which interventions healthcare professionals use to support patients with taking medicines and their perceptions about the effectiveness of those actions.DesignCross-sectional multinational study.SettingOnline survey in Austria, Belgium, England, France, Germany, Hungary, The Netherlands, Poland, Portugal and Switzerland.ParticipantsA total of 3196 healthcare professionals comprising doctors (855), nurses (1047) and pharmacists (1294) currently registered and practising in primary care and community settings.Main outcome measuresPrimary outcome: Responses to the question ‘I ask patients if they have missed any doses of their medication’ for each profession and in each country.Secondary outcome: Responses to 50 items concerning healthcare professional behaviour to support patients with medication-taking for each profession and in each country.ResultsApproximately half of the healthcare professionals in the survey ask patients with long-term conditions whether they have missed any doses of their medication on a regular basis. Pharmacists persistently report that they intervene less than the other two professions to support patients with medicines. No country effects were found for the primary outcome.ConclusionsHealthcare professionals in Europe are limited in the extent to which they intervene to assist patients having long-term conditions with medication adherence. This represents a missed opportunity to support people with prescribed treatment. These conclusions are based on the largest international survey to date of healthcare professionals’ management of medication adherence.