Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly five-fold between 17°C and 37°C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate, suggesting a common dynamical origin. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

Summary Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP‐binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram‐positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR‐type transcriptional repressor LftR, but compounds leading to relief of this repression in wild‐type cells were not known. Using RNA‐Seq and promoter‐lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A – synthesized by the soil‐dwelling Streptomyces aurantiacus – as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic‐producing bacteria in the soil.

The Gram-positive bacterium Listeria monocytogenes occurs ubiquitously in the environment and infects humans upon ingestion. It encodes four PadR-like repressors, out of which LftR has been characterized previously and was shown to control gene expression in response to the antibiotic aurantimycin produced by other environmental bacteria. To better understand the PadR regulons of L . monocytogenes , we performed RNA-sequencing with mutants of the other three repressors LadR, LstR and Lmo0599. We show that LadR is primarily responsible for the regulation of the mdrL gene, encoding an efflux pump, while LstR and Lmo0599 mainly regulate their own operons. The lstR operon contains the lmo0421 gene, encoding a homolog of the RodA/FtsW protein family. However, this protein does not possess such functionality, as we demonstrate here. The lmo0599 operon contains two additional genes coding for the hypothetical trans-membrane proteins lmo0600 and lmo0601 . A striking phenotype of the lmo0599 mutant is its impaired growth at refrigeration temperature. In light of these and other results we suggest that Lmo0599 should be renamed and propose LltR ( l isterial l ow t emperature r egulator) as its new designation. Based on the nature of the PadR target genes we assume that these repressors collectively respond to compounds acting on the cellular envelope.

DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future.IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins.

The bacterium Listeria monocyctogenes ubiquitously occurs in the environment, but can cause severe invasive disease in susceptible individuals when ingested. We recently identified the L. monocytogenes genes lieAB and lftRS, encoding a multi drug resistance ABC transporter and a regulatory module, respectively. These genes jointly mediate resistance against aurantimycin, an antibiotic produced by the soil-dwelling species Streptomyces aurantiacus, and thus contribute to survival of L. monocytogenes in its natural habitat, the soil. Repression of lieAB and lftRS is exceptionally tight, but strongly induced in the presence of aurantimycin. Repression depends on LftR which belongs to subfamily-2 of the PadR-like transcriptional repressors. To better understand this interesting class of transcriptional repressors, we here deduce the LftR operator sequence from a systematic truncation and mutation analysis of the PlieAB promoter. The sequence identified is also present in the PlftRS promoter, but not found elsewhere in the chromosome. Mutational analysis of the putative operator in the PlftRS promoter confirmed its relevance for LftR-dependent repression. The proposed operator sequence was sufficient for DNA binding by LftR in vitro and a mutation in this sequence affected aurantimycin resistance. Our results provide further insights into transcriptional adaptation of an important human pathogen to survive the conditions in its natural reservoir. Importance Listeria monocytogenes is an environmental bacterium that lives in the soil but can infect humans upon ingestion and this can lead to severe invasive disease. Adaptation to these entirely different habitats involves massive reprogramming of transcription. Among the differentially expressed genes is the lieAB operon that encodes a transporter for detoxification of aurantimycin, an antimicrobial compound produced by soil-dwelling competitors. While lieAB is important for survival in the environment, its expression is detrimental during infection. We here identify critical elements in the lieAB promoter and its transcriptional regulator LftR that contribute to habitat-specific expression of the lieAB genes. These results further clarify the molecular mechanisms underlying aurantimycin resistance of L. monocytogenes.