A new T-antigen negative HEK293 cell line with improved AAV productivity.

Viral vectors for gene therapy, such as recombinant adeno-associated viruses, are produced in human embryonic kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene's proprietary HEKExpress,® using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT,® is able to produce high AAV titers, from small to large scale.

RNase J1 and J2 Are Host-Encoded Factors for Plasmid Replication.

Plasmids need to ensure their transmission to both daughter-cells when their host divides, but should at the same time avoid overtaxing their hosts by directing excessive host-resources toward production of plasmid factors. Naturally occurring plasmids have therefore evolved regulatory mechanisms to restrict their copy-number in response to the volume of the cytoplasm. In many plasmid families, copy-number control is mediated by a small plasmid-specified RNA, which is continuously produced and rapidly degraded, to ensure that its concentration is proportional to the current plasmid copy-number. We show here that pSA564 from the RepA_N-family is regulated by a small antisense RNA (RNA1), which, when over-expressed in trans, blocks plasmid replication and cures the bacterial host. The 5' untranslated region (5'UTR) of the plasmid replication initiation gene (repA) potentially forms two mutually exclusive secondary structures, ON and OFF, where the latter both sequesters the repA ribosome binding site and acts as a rho-independent transcriptional terminator. Duplex formation between RNA1 and the 5'UTR shifts the equilibrium to favor the putative OFF-structure, enabling a single small RNA to down-regulate repA expression at both transcriptional and translational levels. We further examine which sequence elements on the antisense RNA and on its 5'UTR target are needed for this regulation. Finally, we identify the host-encoded exoribonucleases RNase J1 and J2 as the enzymes responsible for rapidly degrading the replication-inhibiting section of RNA1. This region accumulates and blocks RepA expression in the absence of either RNase J1 or J2, which are therefore essential host factors for pSA564 replication in Staphylococcus aureus.

Genetic screens reveal novel major and minor players in magnesium homeostasis of Staphylococcus aureus.

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c-di-GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis.

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air-liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.

RNA metabolism in Staphylococcus aureus virulence.

The opportunistic pathogen Staphylococcus aureus encounters a variety of host defence systems depending on its localisation during colonisation in the nares, systemic infections within the body, or persistent infections within cells or embedded in biofilms. To respond rapidly to these different environments, this bacterium has evolved, in its longstanding interaction with animal and human hosts, a variety of mechanisms to fine-tune its gene expression. RNA metabolism, including transcription, processing, translation into proteins and RNA decay, is a central player in this response and might in the future be used to treat this feared pathogen.

An Essential Factor for High Mg2+ Tolerance of Staphylococcus aureus.

Internal bacterial concentration of Mg2+, the most abundant divalent cation in living cells, is estimated to be in the single millimolar range. However, many bacteria will thrive in media with only micromolars of Mg2+, by using a range of intensely studied and highly efficient import mechanisms, as well as in media with very high magnesium concentration, presumably mediated by currently unknown export mechanisms. Staphylococcus aureus has a particularly high Mg2+ tolerance for a pathogen, growing unimpaired in up to 770 mM Mg2+, and we here identify SA0657, a key factor in this tolerance. The predicted domain structure of SA0657 is shared with a large number of proteins in bacteria, archaea and even eukarya, for example CorB from Salmonella and the human CNNM protein family. One of the shared domains, a CBS pair potentially involved in Mg2+ sensing, contains the conserved Glycine326 which we establish to be a key residue for SA0657 function. In light of our findings, we propose the name MpfA, Magnesium Protection Factor A, for SA0657.

Dynamic of bacterial communities attached to lightened phytodetritus.

The effects of singlet oxygen ((1)O2) transfer to bacteria attached on phytodetritus were investigated under laboratory-controlled conditions. For this purpose, a nonaxenic culture of Emiliania huxleyi in late stationary phase was studied for bacterial viability. Our results indicated that only 9 ± 3% of attached bacteria were alive compared to 46 ± 23% for free bacteria in the E. huxleyi culture. Apparently, under conditions of low irradiance (36 W m(-2)), during the culture, the cumulative dose received (22,000 kJ m(-2)) was sufficiently important to induce an efficient (1)O2 transfer to attached bacteria during the senescence of E. huxleyi cells. At this stage, attached bacteria appeared to be dominated by pigmented bacteria (Maribacter, Roseobacter, Roseovarius), which should resist to (1)O2 stress probably due to their high contents of carotenoids. After subsequent irradiation of the culture until fully photodegradation of chlorophyll, DGGE analyses showed that the diversity of bacteria attached to E. huxleyi cells is modified by light. Photooxidative alterations of bacteria were confirmed by the increasing amounts of cis-vaccenic photoproducts (bacterial marker) per bacteria observed during irradiation time. Interestingly, preliminary chemotaxis experiments showed that Shewanella oneidensis considered here as a model of motile bacteria was attracted by phytodetritus producing or not (1)O2. This lack of repulsive effects could explain the high mortality rate of bacteria measured on E. huxleyi cells.

Effects of a sulfonylurea herbicide on the soil bacterial community.

Sulfonylurea herbicides are widely used on a wide range of crops to control weeds. Chevalier® OnePass herbicide is a sulfonylurea herbicide intensively used on cereal crops in Algeria. No information is yet available about the biodegradation of this herbicide or about its effect on the bacterial community of the soil. In this study, we collected an untreated soil sample, and another sample was collected 1 month after treatment with the herbicide. Using a high-resolution melting DNA technique, we have shown that treatment with Chevalier® OnePass herbicide only slightly changed the composition of the whole bacterial community. Two hundred fifty-nine macroscopically different clones were isolated from the untreated and treated soil under both aerobic and microaerobic conditions. The strains were identified by sequencing a conserved fragment of the 16S rRNA gene. The phylogenetic trees constructed using the sequencing results confirmed that the bacterial populations were similar in the two soil samples. Species belonging to the Lysinibacillus, Bacillus, Pseudomonas, and Paenibacillus genera were the most abundant species found. Surprisingly, we found that among ten strains isolated from the treated soil, only six were resistant to the herbicide. Furthermore, bacterial overlay experiments showed that only one resistant strain (related to Stenotrophomonas maltophilia) allowed all the sensitive strains tested to grow in the presence of the herbicide. The other resistant strains allowed only certain sensitive strains to grow. On the basis of these results, we propose that there must be several biodegradation pathways for this sulfonylurea herbicide.

Gram-negative bacteria can also form pellicles.

There is a growing interest in the bacterial pellicle, a biofilm floating at the air-liquid interface. Pellicles have been well studied in the Gram-positive bacterium Bacillus subtilis, but far less in Gram-negative bacteria, where pellicle studies have mostly focused on matrix components rather than on the regulatory cascades involved. Several Gram-negative bacteria, including pathogenic bacteria, have been shown to be able to form a pellicle under static conditions. Here, we summarize the growing body of knowledge about pellicle formation in Gram-negative bacteria, especially about the components of the pellicle matrix. We also propose that the pellicle is a specific biofilm, and that its formation involves particular processes. Since this lifestyle concerns a growing number of bacteria, its properties undoubtedly deserve further investigation.

Aerotaxis governs floating biofilm formation in Shewanella oneidensis.

Floating biofilm, or pellicle, is a biofilm found at the air-liquid interface. Here, we show that pellicle development of the aquatic bacterium Shewanella oneidensis is under the control of the chemotaxis system (Che3), a regulatory system known to pilot planktonic cell motion according to environmental cues. Deletion of the histidine kinase cheA3 or the response regulator cheY3 gene led to a heterogeneous pellicle or to the absence of pellicle respectively. In addition, a non-phosphorylatable CheY3-D56A mutant was unable to promote pellicle formation. Kinetic analysis revealed that pellicle formation occurs in three steps: rapid formation of a thin pellicle evolving into a heterogeneous biofilm and finally into a thick homogeneous biofilm. Depletion of oxygen not only abolished initiation of pellicle formation but also blocked pellicle maturation. This study thus demonstrates an essential role of aerotaxis (chemotaxis towards oxygen) in floating biofilm development in S. oneidensis, and it also reveals that pellicle formation is a step-by-step process.

The chemical-in-µwell: a high-throughput technique for identifying solutes eliciting a chemotactic response in motile bacteria.

Bacteria, and in particular marine bacteria, can be found in environments that are poor in nutrients. To survive, they are able to move toward more favorable niches by a mechanism called chemotaxis, whose first step consists in the detection of substrates by chemoreceptors. We developed a chemotactic assay enabling rapid testing of several hundred different solutes and we identified several molecules eliciting a chemotactic response from two aquatic Shewanella species. We propose that this assay be used for other bacteria to determine the repertoire of chemotactic molecules, generally not clearly elucidated.