The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

Health change awareness and its association with weight loss following bariatric surgery.

OBJECTIVE: Patients' ability to judge health change over time has important clinical implications for treatment, but is understudied in longitudinal contexts with meaningful health change. We assess patients' awareness of health change for 5 years following bariatric surgery, and its association with weight loss. METHOD: Participants were part of the Longitudinal Assessment of Bariatric Surgery (N = 2,027). Perceived health change for each year was assessed by comparing it to self-reports of health on the SF-36 health survey. Participants were categorized as concordant when perceived and actual self-reported health change corresponded, and as discordant when they did not correspond. RESULTS: Year-to-year concordance between perceived and actual self-reported health change occurred less than 50% of the time. Discordance between perceived and actual health was associated with weight loss following surgery. Discordant-positive participants who perceived their health change as more positive than was warranted lost more weight post-surgery and thus had lower body mass index scores than concordant participants. Conversely, discordant-negative participants who perceived their health as worse than what was warranted lost less weight post-surgery and thus had higher body mass index scores. CONCLUSIONS: These results suggest that recollection of past health is generally poor and can be biased by salient factors during recall. Clinicians are advised to use caution when retrospective judgments of health are utilized. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS-StpA Nucleoprotein Filaments in Pathogenic Escherichia coli.

Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH-ops interactions establish long-lived RfaH-EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS-binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS-StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS-StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.

Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments.

Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the H-NS DBD contacts that silence transcription by RNA polymerase, we combined ·OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ∼10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching. Linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contacts.

Principles of mRNA control by human PUM proteins elucidated from multimodal experiments and integrative data analysis.

The human PUF-family proteins, PUM1 and PUM2, posttranscriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3'-UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady-state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a "rulebook" of key contextual features that differentiate functional versus nonfunctional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of High and Low Transcription.

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome.

Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing.

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.

Escherichia coli Lrp Regulates One-Third of the Genome via Direct, Cooperative, and Indirect Routes.

The global regulator Lrp plays a crucial role in regulating metabolism, virulence, and motility in response to environmental conditions. Lrp has previously been shown to activate or repress approximately 10% of the genes in Escherichia coli However, the full spectrum of targets, and how Lrp acts to regulate them, have stymied earlier study. We have combined matched chromatin-immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) under nine physiological conditions to comprehensively map the binding and regulatory activity of Lrp as it directs responses to nutrient abundance. In addition to identifying hundreds of novel Lrp targets, we observe two new global trends, as follows: first, that Lrp will often bind to promoters in a poised position under conditions when it has no regulatory activity to enable combinatorial interactions with other regulators, and second, that nutrient levels induce a global shift in the equilibrium between less-sequence-specific and more-sequence-specific DNA binding. The overall regulatory behavior of Lrp, which as we now show extends to 38% of E. coli genes directly or indirectly under at least one condition, thus arises from the interaction between changes in Lrp binding specificity and cooperative action with other regulators.IMPORTANCE To survive, bacteria such as E. coli must rapidly respond to changing environmental conditions, including nutrient levels. A decrease in nutrient availability causes bacteria to stop rapid replication and enter stationary phase, where they perform limited to no cell division. The E. coli global regulatory protein Lrp has been previously implicated in modulating the expression of genes particularly important at this transition from rapid to slowed growth. Here, we monitor Lrp's DNA binding locations and effect on gene expression under three different nutrient conditions across three growth stages. We find that Lrp's role is even broader than previously suspected and that it appears to interact with many other bacterial regulators to perform its function in a condition-specific manner.

Poor metacognitive awareness of belief change.

When people change beliefs as a result of reading a text, are they aware of these changes? This question was examined for beliefs about spanking as an effective means of discipline. In two experiments, subjects reported beliefs about spanking effectiveness during a prescreening session. In a subsequent experimental session, subjects read a one-sided text that advocated a belief consistent or inconsistent position on the topic. After reading, subjects reported their current beliefs and attempted to recollect their initial beliefs. Subjects reading a belief inconsistent text were more likely to change their beliefs than those who read a belief consistent text. Recollections of initial beliefs tended to be biased in the direction of subjects' current beliefs. In addition, the relationship between the belief consistency of the text read and accuracy of belief recollections was mediated by belief change. This belief memory bias was independent of on-line text processing and comprehension measures, and indicates poor metacognitive awareness of belief change.

Processing and memory of information presented in narrative or expository texts.

BACKGROUND: Previous research suggests that narrative and expository texts differ in the extent to which they prompt students to integrate to-be-learned content with relevant prior knowledge during comprehension. AIMS: We expand on previous research by examining on-line processing and representation in memory of to-be-learned content that is embedded in narrative or expository texts. We are particularly interested in how differences in the use of relevant prior knowledge leads to differences in terms of levels of discourse representation (textbase vs. situation model). SAMPLES: A total of 61 university undergraduates in Expt 1, and 160 in Expt 2. METHODS: In Expt 1, subjects thought out loud while comprehending circulatory system content embedded in a narrative or expository text, followed by free recall of text content. In Expt 2, subjects read silently and completed a sentence recognition task to assess memory. RESULTS: In Expt 1, subjects made more associations to prior knowledge while reading the expository text, and recalled more content. Content recall was also correlated with amount of relevant prior knowledge for subjects who read the expository text but not the narrative text. In Expt 2, subjects reading the expository text (compared to the narrative text) had a weaker textbase representation of the to-be-learned content, but a marginally stronger situation model. CONCLUSIONS: Results suggest that in terms of to-be-learned content, expository texts trigger students to utilize relevant prior knowledge more than narrative texts.

Learning and memory of factual content from narrative and expository text.

BACKGROUND: Research on the presentation of information in narrative versus expository text genres is inconclusive with respect to the question of which is more beneficial for student learning. AIMS: We examine the effect of presenting factual content in either narrative or expository genres on student learning. We also consider relevant prior knowledge and working memory capacity (WMC) as potential mediating variables. SAMPLE: Ninety university undergraduate students. METHODS: Subjects studied circulatory system content embedded in either narrative or expository texts. Prior circulatory system knowledge, knowledge improvement (learning) and free recall were assessed. RESULTS: Learning and recall did not differ as a function of text genre overall, but did interact with prior knowledge. Learning from the narrative and one expository text was optimal at intermediate levels of prior knowledge, with higher knowledge readers benefiting more from the expository text compared with the narrative text. Prior knowledge was positively related to recall for the expository texts, but unrelated for the narrative text. Subjects' WMC did not predict learning or recall. CONCLUSIONS: Results suggest that narrative and expository processing differ with respect to integration of text content with prior knowledge.

Memory for narrative and expository text: independent influences of semantic associations and text organization.

The author examined memory for text in terms of the independent influences of semantic knowledge associations and text organization. Semantic associations were operationalized as the semantic relatedness between individual text concepts and the text as a whole and assessed with latent semantic analysis. The author assessed text organization by simulating comprehension with the construction integration model. Text organization consistently accounted for unique variance in recall. Semantic associations strongly predicted expository recall and predicted narrative recall significantly but to a lesser extent, even when the familiarity of the narrative content was manipulated. Results suggest that prior semantic associations and novel associations in the text structure influence memory independently, and that these influences can be affected by text genre.

Use of latent semantic analysis for predicting psychological phenomena: two issues and proposed solutions.

Latent semantic analysis (LSA) is a computational model of human knowledge representation that approximates semantic relatedness judgments. Two issues are discussed that researchers must attend to when evaluating the utility of LSA for predicting psychological phenomena. First, the role of semantic relatedness in the psychological process of interest must be understood. LSA indices of similarity should then be derived from this theoretical understanding. Second, the knowledge base (semantic space) from which similarity indices are generated must contain 'knowledge' that is appropriate to the task at hand. Proposed solutions are illustrated with data from an experiment in which LSA-based indices were generated from theoretical analysis of the processes involved in understanding two conflicting accounts of a historical event. These indices predict the complexity of subsequent student reasoning about the event, as well as hand-coded predictions generated from think-aloud protocols collected when students were reading the accounts of the event.