bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of the CpsA Protein of Streptococcus agalactiae
Authors: Hanson Brett R. , Runft Donna L. , Streeter Cale , Kumar Abhin , Carion Thomas W. , Neely Melody N. 
Published: January 2012

ABSTRACT

Streptococcal pathogens, such as the group B streptococcus (GBS)
Streptococcus agalactiae
, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the Δ
cpsA
strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the Δ
cpsA
strain, was attenuated in human whole blood. However, the Δ
cpsA
strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.
Global Transcriptional Control by NsrR in Bacillus subtilis
Authors: Kommineni Sushma , Lama Amrita , Popescu Benjamin , Nakano Michiko M. 
Published: January 2012

ABSTRACT

The NO-sensitive NsrR repressor of
Bacillus subtilis
, which carries a [4Fe-4S] cluster, controls transcription of
nasD
and
hmp
(class I regulation) under anaerobic conditions. Here, we describe another class of NsrR regulation (class II regulation) that controls a more diverse collection of genes. Base substitution analysis showed that [4Fe-4S]-NsrR recognizes a partial dyad symmetry within the class I
cis
-acting sites, whereas NO-insensitive interaction of NsrR with an A+T-rich class II regulatory site showed relaxed sequence specificity. Genome-wide transcriptome studies identified genes that are under the control of the class II NsrR regulation. The class II NsrR regulon includes genes controlled by both AbrB and Rok repressors, which also recognize A+T-rich sequences, and by the Fur repressor. Transcription of class II genes was elevated in an
nsrR
mutant during anaerobic fermentative growth with pyruvate. Although NsrR binding to the class II regulatory sites was NO insensitive
in vitro
, transcription of class II genes was moderately induced by NO, which involved reversal of NsrR-dependent repression, suggesting that class II repression is also NO sensitive. In all NsrR-repressed genes tested, the loss of NsrR repressor activity was not sufficient to induce transcription as induction required the ResD response regulator. The ResD-ResE signal transduction system is essential for activation of genes involved in aerobic and anaerobic respiration. This study indicated coordinated regulation between ResD and NsrR and uncovered a new role of ResD and NsrR in transcriptional regulation during anaerobiosis of
B. subtilis
.
Asymmetric Disposal of Individual Protein Aggregates in Escherichia coli, One Aggregate at a Time
Authors: Lloyd-Price Jason , Häkkinen Antti , Kandhavelu Meenakshisundaram , Marques Ines J. , Chowdhury Sharif , Lihavainen Eero , Yli-Harja Olli , Ribeiro Andre S. 
Published: January 2012

ABSTRACTEscherichia colicells employ an asymmetric strategy at division, segregating unwanted substances to older poles, which has been associated with aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites, we tracked individual RNA-MS2-GFP complexes inE. colicells from the time when they were produced. Analyses of the kinetics and brightness of the spots showed that these spots appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased toward one pole, similar to what was observed by previous studies regarding protein aggregates. Additionally, this mechanism was found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes were disposed of, the MS2-GFP tagging molecules alone were not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules and is capable of the accurate classification of molecules for disposal.
Reduced Aeration Affects the Expression of the NorB Efflux Pump of Staphylococcus aureus by Posttranslational Modification of MgrA
Authors: Truong-Bolduc Que Chi , Hsing Liao Chun , Villet Regis , Bolduc Gilles R. , Estabrooks Zoe , Taguezem G Florent , Hooper David C. 
Published: January 2012

ABSTRACT

We previously showed that at acid pH, the transcription of
norB
, encoding the NorB efflux pump, increases due to a reduction in the phosphorylation level of MgrA, which in turn leads to a reduction in bacterial killing by moxifloxacin, a substrate of the NorB efflux pump. In this study, we demonstrated that reduced oxygen levels did not affect the transcript levels of
mgrA
but modified the dimerization of the MgrA protein, which remained mostly in its monomeric form. Under reduced aeration, we also observed a 21.7-fold increase in the
norB
transcript levels after 60 min of growth that contributed to a 4-fold increase in the MICs of moxifloxacin and sparfloxacin for
Staphylococcus aureus
RN6390. The relative proportions of MgrA in monomeric and dimeric forms were altered by treatment with H
2
O
2
, but incubation of purified MgrA with extracts of cells grown under reduced but not normal aeration prevented MgrA from being converted to its dimeric DNA-binding form. This modification was associated with cleavage of a fragment of the dimerization domain of MgrA without change in MgrA phosphorylation and an increase in transcript levels of genes encoding serine proteases in cells incubated at reduced aeration. Taken together, these data suggest that modification of MgrA by proteases underlies the reversal of its repression of
norB
and increased resistance to NorB substrates in response to reduced-aeration conditions, illustrating a third mechanism of posttranslational modification, in addition to oxidation and phosphorylation, that modulates the regulatory activities of MgrA.
Phosphatidylglycerol::Prolipoprotein Diacylglyceryl Transferase (Lgt) of Escherichia coli Has Seven Transmembrane Segments, and Its Essential Residues Are Embedded in the Membrane
Authors: Pailler Jérémy , Aucher Willy , Pires Magali , Buddelmeijer Nienke 
Published: January 2012

ABSTRACTLgt ofEscherichia colicatalyzes the transfer of ansn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins. The enzyme is essential for growth, as demonstrated here by the analysis of anlgtdepletion strain. Cell fractionation demonstrated that Lgt is an inner membrane protein. Its membrane topology was determined by fusing Lgt to β-galactosidase and alkaline phosphatase and bysubstitutedcysteineaccessibilitymethod (SCAM) studies. The data show that Lgt is embedded in the membrane by seven transmembrane segments, that its N terminus faces the periplasm, and that its C terminus faces the cytoplasm. Highly conserved amino acids in Lgt of both Gram-negative and Gram-positive bacteria were identified. Lgt enzymes are characterized by a so-called Lgt signature motif in which four residues are invariant. Ten conserved residues were replaced with alanine, and the activity of these Lgt variants was analyzed by their ability to complement thelgtdepletion strain. Residues Y26, N146, and G154 are absolutely required for Lgt function, and R143, E151, R239, and E243 are important. The results demonstrate that the majority of the essential residues of Lgt are located in the membrane and that the Lgt signature motif faces the periplasm.
Differentiation of Function among the RsbR Paralogs in the General Stress Response of Bacillus subtilis with Regard to Light Perception
Authors: van der Steen Jeroen B. , Ávila-Pérez Marcela , Knippert Doreen , Vreugdenhil Angie , van Alphen Pascal , Hellingwerf Klaas J. 
Published: January 2012

ABSTRACT

The general stress response of
Bacillus subtilis
can be activated by a wide range of signals, including low intensities of visible light. It is regulated by a dedicated σ factor via a complex signal transduction pathway that makes use of stressosomes: hetero-oligomeric complexes that include one or more of the RsbR proteins (RsbRA, RsbRB, RsbRC, and RsbRD). The response to blue light is mediated by the photoreceptor YtvA. We show here which of the four RsbR proteins are necessary for the activation of the σ
B
response by blue light. Experiments performed with single-, double-, and triple-deletion strains in the
rsbR
genes show that RsbRB and RsbRA function antagonistically, with the former being a negative regulator and the latter a positive regulator of the YtvA-dependent light activation of the stress response. A strain with RsbRB as the only RsbR protein is unable to respond to light-activation of σ
B
. Furthermore, RsbRC and RsbRD can replace RsbRA's function only in the absence of RsbRB. This differentiation of function is confined to light stress, since strains with RsbRA or RsbRB as the only RsbR protein behave similarly in our experimental conditions in response to physicochemical stresses. Interestingly, RsbRB's absence is sufficient to result in light activation of the general stress response at wild-type expression levels of
ytvA
, while it was previously reported that YtvA could only activate σ
B
when overproduced, or when cells are supplemented with an additional environmental stress.
Mutational Analyses Reveal Overall Topology and Functional Regions of NilB, a Bacterial Outer Membrane Protein Required for Host Association in a Model of Animal-Microbe Mutualism
Authors: Bhasin Archna , Chaston John M. , Goodrich-Blair Heidi 
Published: January 2012

ABSTRACT

The gammaproteobacterium
Xenorhabdus nematophila
is a mutualistic symbiont that colonizes the intestine of the nematode
Steinernema carpocapsae
.
nilB
(
n
ematode
i
ntestine
l
ocalization) is essential for
X. nematophila
colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by
nilA
and
nilC
, it confers upon noncognate
Xenorhabdus
spp. the ability to colonize
S. carpocapsae
nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.
Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus
Authors: Boutry Céline , Wahl Astrid , Delplace Brigitte , Clippe André , Fontaine Laetitia , Hols Pascal 
Published: January 2012

ABSTRACT

In
Streptococcus thermophilus
, the ComRS regulatory system governs the transcriptional level of
comX
expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation.
In silico
analysis performed on the
S. thermophilus
genome revealed the presence of a homolog of
mecA
(
mecA
St
), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in
Bacillus subtilis
. Using reporter strains and microarray experiments, we showed that MecA
St
represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of
comX
expression but also to be fully dependent on the presence of a functional
comX
gene. This negative control was similarly released in strains deleted for
clpC
,
mecA
, and
clpC-mecA
. Under artificial conditions of
comX
expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in
S. thermophilus
.
The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the
hmuO
and
hrtAB
Promoter Regions
Authors: Burgos Jonathan M. , Schmitt Michael P. 
Published: January 2012

ABSTRACT

Corynebacterium diphtheriae
, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the
hmuO
promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the
hrtAB
genes, which encode an ABC transporter involved in the protection of
C. diphtheriae
from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the
hmuO
and
hrtAB
promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the
hmuO
and
hrtAB
promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the
hmuO
and
hrtAB
promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the
hmuO
promoter region relative to that of wild-type ChrA. DNase I footprint analysis further defined the sequences in the
hmuO
and
hrtAB
promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the
hmuO
promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the
hmuO
promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of
hmuO
and the
hrtAB
genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.
Autoregulation of the Synthesis of the MobM Relaxase Encoded by the Promiscuous Plasmid pMV158
Authors: Lorenzo-Díaz Fabián , Solano-Collado Virtu , Lurz Rudi , Bravo Alicia , Espinosa Manuel 
Published: January 2012

ABSTRACT

The streptococcal promiscuous plasmid pMV158 (5,540 bp) replicates by the rolling-circle mechanism and can be mobilized among a wide number of Gram-positive and -negative bacteria. The plasmid region involved in its conjugative transfer includes the
mobM
gene, which encodes the MobM relaxase, and the
cis
-acting origin of transfer (
oriT
). MobM initiates transfer by cleavage of supercoiled pMV158 DNA at a specific dinucleotide within
oriT
. In the present work, we have performed a detailed transcriptional analysis to assess the role of MobM in the control of its own gene expression. By
in vivo
and
in vitro
approaches, we demonstrated that
mobM
transcription in
Escherichia coli
was mostly initiated from a promoter (
Pmob2
) different from the one (
Pmob1
) used in
Lactococcus lactis
. Whereas promoter
Pmob1
was embedded within the
oriT
sequence, promoter
Pmob2
was placed apart from but adjacent to
oriT
. Further, MobM was able to repress the expression of its own gene from both promoters. Given the promiscuity of pMV158, the organization of the
mobM
promoter region suggests a strategy of the plasmid to cope with different transcription machineries of the hosts it colonizes.
Mammalian-Transmissible H5N1 Influenza: the Dilemma of Dual-Use Research
Authors: Webster Robert G. 
Published: February 2012

ABSTRACT
The National Science Advisory Board for Biosecurity (NSABB)’s recommendation to restrict publication of the details of the generation of mammalian-transmissible H5N1 influenza virus is unprecedented. Dual-use considerations indicated that the potential biosecurity risks of a transmissible H5N1 virus with a possible mortality of 50% in humans outweigh the substantial benefits of open and complete scientific exchange in this case, although the benefits include potential early detection strategies for H5N1 viruses with specific genetic markers and control strategies, including development of antivirals and vaccines. It is argued that both the funding agency (the National Institute of Allergy and Infectious Diseases) and the scientists were responding to societal needs and acted entirely responsibly. These studies usher in a new era for life sciences, compelling the research community to confront important decisions: under what conditions should such research be done? How can the principle of full release of information be balanced with the moral imperative to protect the public health?
Natural Competence Is a Major Mechanism for Horizontal DNA Transfer in the Oral Pathogen Porphyromonas gingivalis
Authors: Tribble Gena D. , Rigney Todd W. , Dao Doan-Hieu V. , Wong Cindy T. , Kerr Jennifer E. , Taylor Brendan E. , Pacha Sara , Kaplan Heidi B. 
Published: February 2012

ABSTRACT

Porphyromonas gingivalis
is a Gram-negative anaerobe that resides exclusively in the human oral cavity. Long-term colonization by
P. gingivalis
requires the bacteria to evade host immune responses while adapting to the changing host physiology and alterations in the composition of the oral microflora. The genetic diversity of
P. gingivalis
appears to reflect the variability of its habitat; however, little is known about the molecular mechanisms generating this diversity. Previously, our research group established that chromosomal DNA transfer occurs between
P. gingivalis
strains. In this study, we examine the role of putative DNA transfer genes in conjugation and transformation and demonstrate that natural competence mediated by
comF
is the dominant form of chromosomal DNA transfer, with transfer by a conjugation-like mechanism playing a minor role. Our results reveal that natural competence mechanisms are present in multiple strains of
P. gingivalis
, and DNA uptake is not sensitive to DNA source or modification status. Furthermore, extracellular DNA was observed for the first time in
P. gingivalis
biofilms and is predicted to be the major DNA source for horizontal transfer and allelic exchange between strains. We propose that exchange of DNA in plaque biofilms by a transformation-like process is of major ecological importance in the survival and persistence of
P. gingivalis
in the challenging oral environment.

IMPORTANCE
P. gingivalis
colonizes the oral cavities of humans worldwide. The long-term persistence of these bacteria can lead to the development of chronic periodontitis and host morbidity associated with tooth loss.
P. gingivalis
is a genetically diverse species, and this variability is believed to contribute to its successful colonization and survival in diverse human hosts, as well as evasion of host immune defenses and immunization strategies. We establish here that natural competence is the major driving force behind
P. gingivalis
DNA exchange and that conjugative DNA transfer plays a minor role. Furthermore, we reveal for the first time the presence of extracellular DNA in
P. gingivalis
biofilms, which is most likely the source of DNA exchanged between strains within dental plaque. These studies expand our understanding of the mechanisms used by this important member of the human oral flora to transition its relationship with the host from a commensal to a pathogenic relationship.