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Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer horizontally to distantly related lineages. Their evolutionary success originates in maximized combined ICE-host fitness trade-offs, but how the ICE impacts on the host metabolism and physiology is poorly understood. Here we investigate global changes in the host genetic network and physiology of Pseudomonas putida with or without an integrated ICE clc , a model ICE widely distributed in proteobacterial genomes. Genome-wide gene expression differences were analyzed by RNA-seq using exponentially growing or stationary phase-restimulated cultures on 3-chlorobenzoate, an aromatic compound metabolizable thanks to specific ICE clc -located genes. We found that the presence of ICE clc imposes a variety of changes in global pathways such as cell cycle and amino acid metabolism, which were more numerous in stationary-restimulated than exponential phase cells. Unexpectedly, ICE clc stimulates cellular motility and leads to more rapid growth on 3-chlorobenzoate than cells carrying only the integrated clc genes. ICE clc also concomitantly activates the P. putida Pspu28-prophage, but this in itself did not provoke measurable fitness effects. ICE clc thus interferes in a number of cellular pathways, inducing both direct benefits as well as indirect costs in P. putida .

Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium’s primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in Escherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

Rasmus Marvig and colleagues report the whole-genome sequencing of 474 longitudinally collected clinical isolates of Pseudomonas aeruginosa sampled from 34 children and young patients with cystic fibrosis. They identify evidence of convergent evolution in 52 genes and suggest pathways involved in within-host adaptation and pathogenesis. Little is known about how within-host evolution compares between genotypically different strains of the same pathogenic species. We sequenced the whole genomes of 474 longitudinally collected clinical isolates of Pseudomonas aeruginosa sampled from 34 children and young individuals with cystic fibrosis. Our analysis of 36 P. aeruginosa lineages identified convergent molecular evolution in 52 genes. This list of genes suggests a role in host adaptation for remodeling of regulatory networks and central metabolism, acquisition of antibiotic resistance and loss of extracellular virulence factors. Furthermore, we find an ordered succession of mutations in key regulatory networks. Accordingly, mutations in downstream transcriptional regulators were contingent upon mutations in upstream regulators, suggesting that remodeling of regulatory networks might be important in adaptation. The characterization of genes involved in host adaptation may help in predicting bacterial evolution in patients with cystic fibrosis and in the design of future intervention strategies.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.

Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli . Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo . Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro . The less efficiently translated native sequences show greatly reduced mRNA levels in vivo . Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli . In-depth analyses of protein expression studies are used to derive a new codon-influence metric that correlates with global protein levels, mRNA levels and mRNA lifetimes in vivo , indicating tight coupling between translation efficiency and mRNA stability; genes redesigned based on these analyses consistently yield high protein expression levels both in vivo and in vitro . Improving protein translation All living organisms use the same set of 64 possible triplet codons to encode 20 amino acids and stop signals during translation of mRNA into protein, but the frequency with which each codon is used varies between organisms. This variation has been thought to be the basis for often poor protein expression when a gene is translated in a heterologous system. John Hunt and colleagues have performed in-depth analysis of protein expression studies to derive a codon-influence metric. When genes are redesigned using this metric, the authors find no effect on their transcription, but much greater translation efficiency. They conclude that in vivo mRNA degradation and translation processes are in competition, and using the metric, protein production can be favoured. This approach may facilitate heterologous gene expression in many biotechnical or experimental settings.

Programmable gene activation enables fine‐tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR‐Cas‐mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR‐Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9‐AsiA, that could activate gene expression by more than 200‐fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9‐AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli . We further identified hundreds of promoters with varying basal expression that could be induced by dCas9‐AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9‐AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR‐based gene regulators. Synopsis An evolved CRISPR‐based bacterial transcription activator system enables programmable and combinatorial gene expression modulation in diverse bacteria. A potent dCas9‐AsiA transcription activator (CasTA) is identified and evolved using a bacterial CRISPRa screening platform. A wide regulatory region can be targeted by CasTA, yielding increase gene expression by > 100‐fold. A synthetic CasTA‐inducible promoter library is developed. CasTA functions in multiple bacterial species and enables bacterial gene activation and repression. Graphical Abstract An evolved CRISPR‐based bacterial transcription activator system enables programmable and combinatorial gene expression modulation in diverse bacteria.

Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen. This study shows that glutathione, a ubiquitous antioxidant, is also a critical signalling molecule that allosterically activates the master virulence regulator in the intracellular pathogen  Listeria monocytogenes . Glutathione signals promote Listeria pathogenicity To successfully colonize their hosts, intracellular pathogens must be able to sense their environment and modulate virulence gene expression. For instance, when Listeria monocytogenes infects host cells, it remodels its transcriptional program through activation of the master regulator PrfA. Previous work has suggested that PrfA is allosterically regulated by a small molecule activator, specific to the host intracellular environment, but the identity of this small molecule has proven elusive. Here Daniel Portnoy and colleagues show that bacterial and host-derived glutathione is essential for L. monocytogenes pathogenesis, but not via its canonical role in redox homeostasis. Rather, glutathione activates PrfA by acting as the previously predicted allosteric modulator.