Summary Host‐encoded functions that regulate the transfer operon (tra) in the virulence plasmid of Salmonella enterica (pSLT) were identified with a genetic screen. Mutations that decreased tra operon expression mapped in the lrp gene, which encodes the leucine‐responsive regulatory protein (Lrp). Reduced tra operon expression in an Lrp− background is caused by lowered transcription of the traJ gene, which encodes a transcriptional activator of the tra operon. Gel retardation assays indicated that Lrp binds a DNA region upstream of the traJ promoter. Deletion of the Lrp binding site resulted in lowered and Lrp‐independent traJ transcription. Conjugal transfer of pSLT decreased 50‐fold in a Lrp− background. When a FinO− derivative of pSLT was used, conjugal transfer from an Lrp− donor decreased 1000‐fold. Mutations that derepressed tra operon expression mapped in dam, the gene encoding Dam methyltransferase. Expression of the tra operon and conjugal transfer remain repressed in an Lrp− Dam− background. These observations support the model that Lrp acts as a conjugation activator by promoting traJ transcription, whereas Dam methylation acts as a conjugation repressor by activating FinP RNA synthesis. This dual control of conjugal transfer may also operate in other F‐like plasmids such as F and R100.

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

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Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria. Here we have developed an inducible autolysis system based in the lysis operon of the lambda phage that, in response to anhydrotetracycline, lysates Salmonella thus releasing its content. The system was combined with a salicylate cascade system that allows efficient production of therapeutic molecules in response to aspirin and with a sifA mutation that liberates bacteria from the vacuoles to a cytosolic location. The combination of these three elements makes this strain a putative powerful instrument in cancer treatment. We have used this engineered strain for the intracellular production and delivery of Cp53 peptide. The engineered strain is able to sequentially produce and release the cytotoxic peptide while proliferating inside tumour cells, thus inducing host cell death. Our results show that temporal separation of protein production from protein release is essential to efficiently kill tumour cells. The combined system is a further step in the engineering of more efficient bacteria for cancer therapy.

DNA adenine methylase (DamThe F sex factor and its relatives have served as a paradigm of bacterial conjugation for six decades (62). Early studies carried out with F paved the way for the discovery of plasmids in other bacterial genera. For instance, a plasmid initially called "cryptic" and later found to contain virulence genes was discovered in the standard strain LT2 of Salmonella enterica serovar Typhimurium (51,52). Virulence plasmids are also found in other strains of S. enterica serovar Typhimurium, such as the mouse virulent strains SL1344 and 14028, and in other host-adapted serotypes of S. enterica except the humanadapted serovar Typhi (46).The virulence plasmid of S. enterica strain LT2 (pSLT) is self-transmissible (1). Unlike F, pSLT undergoes conjugation at low frequencies (1,5), and the genetic mechanism that maintains conjugal repression can cross-regulate F transfer (48,51,59). Repression of mating is the norm for most F-like plasmids, and it relies on the so-called fertility inhibition system (17). Transcription of the main transfer operon (tra), which encodes functions for pilus synthesis and DNA processing during conjugal transfer, requires activation by the TraJ protein, encoded on a nearby monocistronic transcriptional unit (18,19,41). Synthesis of the TraJ transcriptional activator is prevented by a small, untranslated RNA encoded by an overlapping gene, finP (20,41). Pairing between FinP RNA and the traJ mRNA leader triggers degradation of the RNA duplex by RNase III (29), a phenomenon reminiscent of the RNA interference mechanism described in eukaryotes (38). FinP RNA is a short-lived molecule unless it is stabilized by the FinO protein (30). FinO protects FinP RNA from degradation by RNase E (29) and acts as an RNA chaperone that facilitates RNA-RNA interactions (2). The F sex factor is a FinO Ϫ mutant (8), and the ability of pSLT to repress F transfer relies on providing trans-acting FinO protein (48). In certain F-like plasmids, activation of the main tra promoter, traY, is also activated by transcription of the plasmid-borne gene traM. Transcripts originating at the traM promoter appear to enter the neighboring traJ gene, thereby providing a two-cistron mRNA that can unbalance the finP/traJ transcriptional ratio and yield TraJ product for the activation of the traY promoter (11).Besides these plasmid-encoded regulators of tra operon expression, chromosomal regulators of mating are also known (39). The list includes the transcriptional regulators ArcA (55, 57) and CRP (53), the nucleoid protein H-NS (54, 61), the leucine-responsive regulatory protein (5), and DNA adenine methylase (Dam) methylation (59). A postranscriptional control mechanism acting on the TraJ product has been also described (23, 50).Regulation of conjugal transfer by Dam methylation occurs in F (59), pSLT (5), and R100 (7). In all cases, Dam methylation acts as a repressor of mating, and the increase in conjugation frequency detected in Dam Ϫ donors varies from plasmid to plasmid (5,7,59 Downloaded fromIn this stud...

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

Summary The presence of pharmaceutical compounds in waters and soils is of particular concern because these compounds can be biologically active, even at environmental concentrations. Most pharmaceutical contaminants result from inefficient removal of these compounds during wastewater treatment. Although microorganisms able to biodegrade pharmaceuticals compounds have been described, the isolation and characterization of new bacterial strains capable of degrading drugs remain important to improve the removal of this pollutant. In this work, we describe the Sphingomonas wittichii strain MPO218 as able to use ibuprofen as the sole carbon and energy source. The genome of MPO218 consists of a circular chromosome and two circular plasmids. Our analysis shows that the largest plasmid, named pIBU218, is conjugative and can horizontally transfer the capability of growing on ibuprofen after conjugation with another related bacterium, Sphingopyxis granuli TFA. This plasmid appears to be unstable since it undergoes different deletions in absence of selection when growth on ibuprofen is not selected. This is the first described example of a natural and conjugative plasmid that enables growth on ibuprofen and is another example of how horizontal gene transfer plays a crucial role in the evolution of bacteria.

The virulence plasmid of Salmonella enterica (pSLT) is an F-like conjugative plasmid. High rates of pSLT transfer occur in the mammalian gut, a microaerobic environment. In this study, we describe genetic screens for host-encoded activators and repressors of the transfer operon (tra) of pSLT. We show that the transcription factor ArcA is an activator of conjugation, especially under microaerobiosis. In turn, succinate dehydrogenase (SdhABCD) is a repressor of mating in aerobiosis. ArcA binds upstream of the main tra promoter (p traY ) and activates tra transcription, as previously described in F, R1, and R100. In the absence of ArcA, transfer of pSLT decreased 7-fold in aerobiosis and .100-fold in microaerobiosis. In aerobiosis, ArcA activates the traY promoter in an ArcB-independent manner, as described in other F-like plasmids. In microaerobiosis, however, the ArcB sensor is necessary for activation of p traY . Lack of Sdh causes a .20-fold increase in pSLT transfer in aerobiosis, but has little effect under microaerobiosis. Sdh inhibits conjugal transfer by reducing traJ transcription, probably in an indirect manner. In turn, the sdhCDAB operon is repressed by the ArcAB system under microaerobiosis. Hence, the ArcAB twocomponent system of S. enterica stimulates pSLT transfer under microaerobiosis by two concerted actions: activation of the tra operon and repression of the sdhCDAB operon.