Regulation of Gene Expression of phiEco32-like Bacteriophage 7-11

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity  subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11  subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36  factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.

Transcriptomic Fingerprint of Bacterial Infection in Lower Extremity Ulcers

Clinicians and researchers utilize subjective classification systems based on clinical parameters to stratify lower extremity ulcer infections for treatment and research. This study compared clinical infection classifications (mild to severe) of lower extremity ulcers (n = 44) with transcriptomic profiles and direct measurement of bacterial RNA signatures by RNA-sequencing. Samples demonstrating similar transcriptomes were clustered and characterized by transcriptomic fingerprint. Clinical infection severity did not explain the major sources of variability among the samples and samples with the same clinical classification demonstrated high inter-sample variability. High proportions of bacterial RNA, however, resulted in a strong effect on transcription and increased expression of genes associated with immune response and inflammation. K-means clustering identified two clusters of samples, one of which contained all of the samples with high levels of bacterial RNA. A support vector classifier identified a fingerprint of 20 genes, including immune-associated genes such as CXCL8, GADD45B, and HILPDA, which accurately identified samples with signs of infection via cross-validation. This suggests that stratification of infection states based on a transcriptomic fingerprint may be a useful tool for studying host-bacterial interactions in these ulcers, as well as an objective classification method to identify the severity of infection.

First isolation of Arcanobacterium pinnipediorum from a grey seal pup (Halichoerus grypus) in the UK

In the present study, a single Arcanobacterium (A.) pinnipediorum strain isolated from discharge of a jaw swelling of a grey seal pup (Halichoerus grypus) in England, UK, was identified. This strain was further characterized by phenotypical investigations, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), by Fourier transform infrared spectroscopy (FT-IR), and genotypically by sequencing the 16S rRNA gene and the genes gap encoding glyceraldehyde 3-phosphate dehydrogenase, tuf encoding elongation factor tu, and rpoB encoding the β subunit of bacterial RNA polymerase. The present study gives a first detailed characterization of the species A. pinnipediorum from a grey seal in the UK. However, the route of infection of the grey seal with the bacterial pathogen remains unclear.

NUDT2 initiates viral RNA degradation by removal of 5′-phosphates

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.

Direct RNA nanopore sequencing of Pseudomonas aeruginosa clone C transcriptomes

The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phase of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules followed by similar proportions of mRNA transcripts and non-coding RNAs. Both platforms detected similar proportions of uncharged tRNAs and 29 yet undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA Arg , and the tRNA Arg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct non-overlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range of a few hundred to thousands of bases and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet undescribed RNA molecules and an unexpected composition of the pools of tRNAs, sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of ribosomal RNAs, enzymatic reverse transcription and the fragmentation, size selection and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that – if replicated in other species – will change our view of the bacterial RNA world. The discovery of sense – antisense pairs of tmRNA, tRNAs and mRNAs indicates a further and unknown level of gene regulation in bacteria.

Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLink® RNA Mini Kit and Basic Laboratory Instrument

RNA extraction is an important process before gene expression assessment at the transcriptomic level. RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments. In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLink® RNA Mini Kit. The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. The results of RNA extraction were visualized using agarose gel electrophoresis. These results indicate that bacterial RNA extraction can be performed using the PureLink® RNA Mini Kit even with inexpensive basic laboratory instruments.

Novel RNA Extraction Method for Dual RNA-seq Analysis of Pathogen and Host in the Early Stages of Yersinia pestis Pulmonary Infection

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.