Transcriptional and post-transcriptional mechanisms modulate cyclopropane fatty acid synthase through small RNAs in Escherichia coli.

The small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases the conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The regulators that control RydC synthesis had not previously been identified. In this study, we identify a GntR-family transcription factor, YieP, that represses rydC transcription. YieP positively autoregulates its own transcription and indirectly regulates cfa through RydC. We further identify additional sRNA regulatory inputs that contribute to the control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link the control of membrane biophysical properties to multiple environmental signals. IMPORTANCE: Bacteria experience many environmental stresses that challenge their membrane integrity. To withstand these challenges, bacteria sense what stress is occurring and mount a response that protects membranes. Previous work documented the important roles of small RNA (sRNA) regulators in membrane stress responses. One sRNA, RydC, helps cells cope with membrane-disrupting stresses by promoting changes in the types of lipids incorporated into membranes. In this study, we identified a regulator, YieP, that controls when RydC is produced and additional sRNA regulators that modulate YieP levels and RydC activity. These findings illuminate a complex regulatory network that helps bacteria sense and respond to membrane stress.

A two-variant model of SARS-COV-2 transmission: estimating the characteristics of a newly emerging strain.

BACKGROUND: The Covid-19 pandemic has been characterized by the emergence of novel SARS-CoV-2 variants, each with distinct properties influencing transmission dynamics, immune escape, and virulence, which, in turn, influence their impact on local populations. Swift analysis of the properties of newly emerged variants is essential in the initial days and weeks to enhance readiness and facilitate the scaling of clinical and public health system responses. METHODS: This paper introduces a two-variant metapopulation compartmental model of disease transmission to simulate the dynamics of disease transmission during a period of transition to a newly dominant strain. Leveraging novel S-gene dropout analysis data and genomic sequencing data, combined with confirmed Covid-19 case data, we estimate the epidemiological characteristics of the Omicron variant, which replaced the Delta variant in late 2021 in Philadelphia, PA. We utilized a grid-search method to identify plausible combinations of model parameters, followed by an ensemble adjustment Kalman filter for parameter inference. RESULTS: The model successfully estimated key epidemiological parameters; we estimated the ascertainment rate of 0.22 (95% credible interval 0.15-0.29) and transmission rate of 5.0 (95% CI 2.4-6.6) for the Omicron variant. CONCLUSIONS: The study demonstrates the potential for this model-inference framework to provide real-time insights during the emergence of novel variants, aiding in timely public health responses.

Bacterial Cyclopropane Fatty Acid Synthase mRNA Is Targeted by Activating and Repressing Small RNAs.

Altering membrane protein and lipid composition is an important strategy for maintaining membrane integrity during environmental stress. Many bacterial small RNAs (sRNAs) control membrane protein production, but sRNA-mediated regulation of membrane fatty acid composition is less well understood. The sRNA RydC was previously shown to stabilize cfa (cyclopropane fatty acid synthase) mRNA, resulting in higher levels of cyclopropane fatty acids in the cell membrane. Here, we report that additional sRNAs, ArrS and CpxQ, also directly regulate cfa posttranscriptionally. RydC and ArrS act through masking an RNase E cleavage site in the cfa mRNA 5' untranslated region (UTR), and both sRNAs posttranscriptionally activate cfa In contrast, CpxQ binds to a different site in the cfa mRNA 5' UTR and represses cfa expression. Alteration of membrane lipid composition is a key mechanism for bacteria to survive low-pH environments, and we show that cfa translation increases in an sRNA-dependent manner when cells are subjected to mild acid stress. This work suggests an important role for sRNAs in the acid stress response through regulation of cfa mRNA.IMPORTANCE Small RNAs (sRNAs) in bacteria are abundant and play important roles in posttranscriptional regulation of gene expression, particularly under stress conditions. Some mRNAs are targets for regulation by multiple sRNAs, each responding to different environmental signals. Uncovering the regulatory mechanisms governing sRNA-mRNA interactions and the relevant conditions for these interactions is an ongoing challenge. In this study, we discovered that multiple sRNAs control membrane lipid composition by regulating stability of a single mRNA target. The sRNA-dependent regulation occurred in response to changing pH and was important for cell viability under acid stress conditions. This work reveals yet another aspect of bacterial physiology controlled at the posttranscriptional level by sRNA regulators.

Pre-epidemic evolution of the MRSA USA300 clade and a molecular key for classification.

Introduction: USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Methods: Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Results: Phylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

Variable staphyloxanthin production by Staphylococcus aureus drives strain-dependent effects on diabetic wound-healing outcomes.

Strain-level variation in Staphylococcus aureus is a factor that contributes to disease burden and clinical outcomes in skin disorders and chronic wounds. However, the microbial mechanisms that drive these variable host responses are poorly understood. To identify mechanisms underlying strain-specific outcomes, we perform high-throughput phenotyping screens on S. aureus isolates cultured from diabetic foot ulcers. Isolates from non-healing wounds produce more staphyloxanthin, a cell membrane pigment. In murine diabetic wounds, staphyloxanthin-producing isolates delay wound closure significantly compared with staphyloxanthin-deficient isolates. Staphyloxanthin promotes resistance to oxidative stress and enhances bacterial survival in neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes reveals a mutation in the sigma B operon, resulting in marked differences in stress response gene expression. Our work illustrates a framework to identify traits that underlie strain-level variation in disease burden and suggests more precise targets for therapeutic intervention in S. aureus-positive wounds.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 Isolates.

We report the genome of a B.1.1.7+E484K severe acute respiratory syndrome coronavirus 2 from Southeastern Pennsylvania and compare it with all high-coverage B.1.1.7+E484K genomes (n = 235) available. Analyses showed the existence of at least 4 distinct clades of this variant circulating in the United States and the possibility of at least 59 independent acquisitions of the E484K mutation.

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote "escape" from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Dual-Function RNAs.

Bacteria are known to use RNA, either as mRNAs encoding proteins or as noncoding small RNAs (sRNAs), to regulate numerous biological processes. However, a few sRNAs have two functions: they act as base-pairing RNAs and encode a small protein with additional regulatory functions. Thus, these so called "dual-function" sRNAs can serve as both a riboregulator and an mRNA. In some cases, these two functions can act independently within the same pathway, while in other cases, the base-pairing function and protein function act in different pathways. Here, we discuss the five known dual-function sRNAs-SgrS from enteric species, RNAIII and Psm-mec from Staphylococcus aureus, Pel RNA from Streptococcus pyogenes, and SR1 from Bacillus subtilis-and review their mechanisms of action and roles in regulating diverse biological processes. We also discuss the prospect of finding additional dual-function sRNAs and future challenges in studying the overlap and competition between the functions.

Light intensity impacts the production of biofuel intermediates in Heterosigma akashiwo growing on simulated flue gas containing carbon dioxide and nitric oxide.

As a potential biofuel feedstock, the marine microalga, Heterosigma akashiwo, accumulates significant lipids, is capable of long-term growth in outdoor photobioreactors, and is an excellent candidate for the bioremediation of industrial emissions. Here, we evaluated resource partitioning in H. akashiwo growing on a CO2 and NO gas mixture under three light intensities: 160, 560, or 1200µmolquantam(-2)s(-1). Light levels had no effect on growth; however, cultures in high light accumulated 2.3-fold more carbohydrates and 17% fewer lipids. Light levels did not affect the percentage of saturated fatty acids, but mono-unsaturates increased by 6% and poly-unsaturates decreased by 12% in high light. The fatty acid profiles reported here suggest that H. akashiwo is a good candidate for the production of neutral lipids for biodiesel and also omega-3 fatty acids, and that the quality of biodiesel acquired from feedstocks grown under fluctuating light conditions would be relatively stable.