While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli ...transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser.
Many bacterial small RNAs (sRNAs) regulate gene expression through base-pairing with mRNAs, and it has been assumed that these sRNAs act solely by this one mechanism. Here we report that the ...multicellular adhesive (McaS) sRNA of Escherichia coli uniquely acts by two different mechanisms: base-pairing and protein titration. Previous work established that McaS base pairs with the mRNAs encoding master transcription regulators of curli and flagella synthesis, respectively, resulting in down-regulation and up-regulation of these important cell surface structures. In this study, we demonstrate that McaS activates synthesis of the exopolysaccharide β-1,6 N-acetyl-D-glucosamine (PGA) by binding the global RNA-binding protein CsrA, a negative regulator of pgaA translation. The McaS RNA bears at least two CsrA-binding sequences, and inactivation of these sites compromises CsrA binding, PGA regulation, and biofilm formation. Moreover, ectopic McaS expression leads to induction of two additional CsrA-repressed genes encoding diguanylate cyclases. Collectively, our study shows that McaS is a dual-function sRNA with roles in the two major post-transcriptional regulons controlled by the RNA-binding proteins Hfq and CsrA.
The Gram-positive bacterium
Listeria monocytogenes
is the causative agent of the foodborne disease listeriosis, one of the deadliest bacterial infections known. In order to cause disease,
L
.
...monocytogenes
must properly coordinate its metabolic and virulence programs in response to rapidly changing environments within the host. However, the mechanisms by which
L
.
monocytogenes
senses and adapts to the many stressors encountered as it transits through the gastrointestinal (GI) tract and disseminates to peripheral organs are not well understood. In this study, we investigated the role of the redox-responsive transcriptional regulator Rex in
L
.
monocytogenes
growth and pathogenesis. Rex is a conserved canonical transcriptional repressor that monitors the intracellular redox state of the cell by sensing the ratio of reduced and oxidized nicotinamide adenine dinucleotides (NADH and NAD
+
, respectively). Here, we demonstrated that
L
.
monocytogenes
Rex represses fermentative metabolism and is therefore required for optimal growth in the presence of oxygen. We also show that
in vitro
, Rex represses the production of virulence factors required for survival and invasion of the GI tract, as a strain lacking
rex
was more resistant to acidified bile and invaded host cells better than wild type. Consistent with these results, Rex was dispensable for colonizing the GI tract and disseminating to peripheral organs in an oral listeriosis model of infection. However, Rex-dependent regulation was required for colonizing the spleen and liver, and
L
.
monocytogenes
lacking the Rex repressor were nearly sterilized from the gallbladder. Taken together, these results demonstrated that Rex functions as a repressor of fermentative metabolism and suggests a role for Rex-dependent regulation in
L
.
monocytogenes
pathogenesis. Importantly, the gallbladder is the bacterial reservoir during listeriosis, and our data suggest redox sensing and Rex-dependent regulation are necessary for bacterial survival and replication in this organ.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised individuals. Macrophages play a dual role during Lm infection by ...both promoting dissemination of Lm from the gastrointestinal tract and limiting bacterial growth upon immune activation. Despite the relevance of macrophages to Lm infection, the mechanisms underlying phagocytosis of Lm by macrophages are not well understood. To identify host factors important for Lm infection of macrophages, we performed an unbiased CRISPR/Cas9 screen which revealed pathways that are specific to phagocytosis of Lm and those that are required for internalization of bacteria generally. Specifically, we discovered the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, we found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, we show that PTEN-dependent phagocytosis is important for host protection during oral Lm infection. Overall, this study provides a comprehensive identification of macrophage factors involved in regulating Lm uptake and characterizes the function of one factor, PTEN, during Lm infection in vitro and in vivo. Importantly, these results demonstrate a role for opsonin-independent phagocytosis in Lm pathogenesis and suggest that macrophages play a primarily protective role during foodborne listeriosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence ...can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multidrug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modelling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase β-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.
Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) ...generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Though studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen
induces many genes in response to 4-HNE exposure. A component of the
4-HNE response is the expression of the genes
and
deemed
and
(reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on
bacterial burdens during murine or tissue culture infection. However, heterologous expression of
in
significantly increased bacterial resistance to 4-HNE
and promoted bacterial survival following phagocytosis by murine macrophages in an ROS dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in
but are sufficient to confer resistance to an otherwise sensitive organism
and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.
Context.
We present results from the first recorded stellar occultation by the large trans-Neptunian object (174567) Varda that was observed on September 10, 2018. Varda belongs to the ...high-inclination dynamically excited population, and has a satellite, Ilmarë, which is half the size of Varda.
Aims.
We determine the size and albedo of Varda and constrain its 3D shape and density.
Methods.
Thirteen different sites in the USA monitored the event, five of which detected an occultation by the main body. A best-fitting ellipse to the occultation chords provides the instantaneous limb of the body, from which the geometric albedo is computed. The size and shape of Varda are evaluated, and its bulk density is constrained using Varda’s mass as is known from previous works.
Results.
The best-fitting elliptical limb has semi-major (equatorial) axis of (383 ± 3) km and an apparent oblateness of 0.066 ± 0.047, corresponding to an apparent area-equivalent radius
R
′
equiv
= (370±7) km and geometric albedo
p
v
= 0.099 ± 0.002 assuming a visual absolute magnitude
H
V
= 3.81 ± 0.01. Using three possible rotational periods for the body (4.76, 5.91, and 7.87 h), we derive corresponding MacLaurin solutions. Furthermore, given the low-amplitude (0.06 ± 0.01) mag of the single-peaked rotational light-curve for the aforementioned periods, we consider the double periods. For the 5.91 h period (the most probable) and its double (11.82 h), we find bulk densities and true oblateness of
ρ
= (1.78 ± 0.06) g cm
−3
,
ɛ
= 0.235 ± 0.050, and
ρ
= (1.23 ± 0.04) g cm
−3
,
ɛ
= 0.080 ± 0.049. However, it must be noted that the other solutions cannot be excluded just yet.
Summary
In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base‐pairing with mRNA targets to affect protein translation ...or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS‐related protein. We show that McaS is a ∼ 95 nt transcript whose expression increases over growth, peaking in early‐to‐mid stationary phase, or when glucose is limiting. McaS uses three discrete single‐stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly β‐1,6‐N‐acetyl‐d‐glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.
Efforts to battle antimicrobial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. ...Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an “evolvability factor” that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd’s role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with “anti-evolution” drugs)—in particular, Mfd—providing an unexplored route toward battling the AMR crisis.
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•The bacterial transcription-coupled repair (TCR) factor Mfd promotes mutagenesis•Mfd-driven mutagenesis accelerates the evolution of antimicrobial resistance (AMR)•The rapid evolution of AMR requires Mfd’s interaction with RpoB and UvrA•Mfd may be an ideal target for “anti-evolution” drugs that inhibit AMR development
Bacterial evolution drives antimicrobial resistance (AMR) development. We identify the protein Mfd as a highly conserved “evolvability factor” that increases mutagenesis and the capacity of bacteria to evolve antibiotic resistance. We propose inhibiting the activity of evolvability factors through “anti-evolution” drugs during antibiotic treatment to ameliorate the global AMR crisis.
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