Disa L. Hammarlöf

An Infection-Relevant Transcriptomic Compendium for Salmonella enterica Serovar Typhimurium

Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community.

Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu

Salmonella enterica with mutant EF‐Tu (Gln125Arg) has a low level of EF‐Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n= 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n= 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N‐terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild‐type levels in the tuf‐rne double mutant. In contrast, the half‐lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon‐specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF‐Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide

Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non‐typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non‐iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O‐antigen modification. GtrCBTP1 is essential for maintaining O‐antigen length in isolate D23580, since a gtrBTP1 mutant yields a short O‐antigen. This phenotype can be complemented by gtrCBTP1 or very closely related gtrC genes. The short O‐antigen of the gtrBTP1 mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O‐antigen cleavage. Expression of the gtrCBTP1 gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrCBTP1 expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrCBTP1 mediates modification of the BTP1 phage O‐antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O‐antigen co‐receptor.

Characterization of the Prophage Repertoire of African Salmonella Typhimurium ST313 Reveals High Levels of Spontaneous Induction of Novel Phage BTP1

In the past 30 years, Salmonella bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of an estimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of Salmonella Typhimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2D23580, ST64BD23580, Gifsy-1D23580, and BTP5. We show that common S. Typhimurium prophages Gifsy-2, Gifsy-1, and ST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species “Salmonella virus BTP1”, belonging to the P22virus genus. Surprisingly, ∼109 BTP1 virus particles per ml were detected in the supernatant of non-induced, stationary-phase cultures of strain D23580, representing the highest spontaneously induced phage titer so far reported for a bacterial prophage. High spontaneous induction is shown to be an intrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around 0.2% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophages in epidemic S. Typhimurium ST313.

Turnover of mRNAs is one of the essential functions of RNase E

RNase E is an essential bacterial endoribonuclease with a central role in processing tRNAs and rRNA, and turning over mRNAs. Previous studies in strains carrying mutations in the rne structural gene have shown that tRNA processing is likely to be an essential function of RNase E but have not determined whether mRNA turnover is also an essential function. To address this we selected extragenic suppressors of temperature‐sensitive mutations in rne that cause a large increase in mRNA half‐life at the non‐permissive temperature. Fifteen suppressors were mapped to three different loci: relBE (toxin‐antitoxin system); vacB (RNase R); and rpsA (ribosomal protein S1). Each suppressor class has the potential to interact with mRNA and each restores wild‐type levels of mRNA turnover but does not reverse the minor defects in tRNA and rRNA processing. RelE toxin is especially interesting because its only known activity is to cleave mRNAs in the ribosomal A‐site. The relBE suppressor mutations increase transcription of relE, and controlled overexpression of RelE alone was sufficient to suppress the rne ts phenotype. Suppression increased turnover of some major mRNAs (tufA, ompA) but not all mRNAs. We propose that turnover of some mRNAs is one of the essential functions of RNase E.

Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu

Transcription and translation of mRNAs are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.

Temperature-sensitive mutants of RNase E in Salmonella enterica.

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella

Significance Invasive nontyphoidal Salmonella disease is a major and previously neglected tropical disease responsible for an estimated ∼390,000 deaths per year in Africa, largely caused by a variant of Salmonella Typhimurium called ST313. Despite the availability of >100,000 Salmonella genomes, it has proven challenging to associate individual SNPs with pathogenic traits of this dangerous bacterium. Here, we used a transcriptomic strategy to identify a single-nucleotide change in a promoter region responsible for crucial phenotypic differences of African S. Typhimurium. Our findings show that a noncoding nucleotide of the bacterial genome can have a profound effect upon the pathogenesis of infectious disease. Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S. Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.

The FUN of identifying gene function in bacterial pathogens; insights from Salmonella functional genomics.

The availability of thousands of genome sequences of bacterial pathogens poses a particular challenge because each genome contains hundreds of genes of unknown function (FUN). How can we easily discover which FUN genes encode important virulence factors? One solution is to combine two different functional genomic approaches. First, transcriptomics identifies bacterial FUN genes that show differential expression during the process of mammalian infection. Second, global mutagenesis identifies individual FUN genes that the pathogen requires to cause disease. The intersection of these datasets can reveal a small set of candidate genes most likely to encode novel virulence attributes. We demonstrate this approach with the Salmonella infection model, and propose that a similar strategy could be used for other bacterial pathogens.

Contact‐dependent growth inhibition induces high levels of antibiotic‐tolerant persister cells in clonal bacterial populations

Bacterial populations can use bet‐hedging strategies to cope with rapidly changing environments. One example is non‐growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact‐dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI‐mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon‐mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density‐dependent bet‐hedging strategy, where the fraction of non‐growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.