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In this study, we assessed the potential of bacteriocins and their

The continuous emergence of microbial resistance to our antibiotic arsenal is widely becoming recognized as an imminent threat to global human health. Bacteriocins are antimicrobial peptides currently under consideration as real alternatives or complements to common antibiotics. These peptides have been much studied, novel bacteriocins are regularly reported and several genomic databases on these peptides are currently updated. Despite this, to our knowledge, a physical collection of bacteriocins that would allow testing and comparing them for different applications does not exist. Rapid advances in synthetic biology in combination with cell-free protein synthesis technologies offer great potential for fast protein production. Based on the amino acid sequences of the mature peptide available in different databases, we have built a bacteriocin gene library, called PARAGEN 1.0, containing all the genetic elements required for in vitro cell-free peptide synthesis. Using PARAGEN 1.0 and a commercial kit for cell-free protein synthesis we have produced 164 different bacteriocins. Of the bacteriocins synthesized, 54% have shown antimicrobial activity against at least one of the indicator strains tested, including Gram-positive and Gram-negative bacteria representing commonly used lab strains, industrially relevant microorganisms, and known pathogens. This bacteriocin collection represents a streamlined pipeline for selection, production, and screening of bacteriocins as well as a reservoir of ready-to-use antimicrobials against virtually any class of relevant bacteria.

It has been shown previously that female mice homozygous for an alpha-fetoprotein (AFP) null allele are sterile as a result of anovulation, probably due to a defect in the hypothalamic-pituitary axis. Here we show that these female mice exhibit specific anomalies in the expression of numerous genes in the pituitary, including genes involved in the gonadotropin-releasing hormone pathway, which are underexpressed. In the hypothalamus, the gonadotropin-releasing hormone gene, Gnrh1, was also found to be down-regulated. However, pituitary gene expression could be normalized and fertility could be rescued by blocking prenatal estrogen synthesis using an aromatase inhibitor. These results show that AFP protects the developing female brain from the adverse effects of prenatal estrogen exposure and clarify a long-running debate on the role of this fetal protein in brain sexual differentiation.

The alpha-fetoprotein gene ( Afp ) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

Epitope tagging simplifies detection, characterization and purification of proteins. Gene fusion to combine the coding region of a well-characterized epitope with the coding region for a protein of interest generally requires several subcloning steps. Alternatively, a PCR strategy can be used to generate such a chimeric gene. In addition to its simplicity, this approach allows one to limit the size of the multiple cloning sites present in conventional expression vectors, thus reducing the introduction of artifactual amino-acid sequences into the fused protein. In this communication, we describe new vectors that allow PCR cloning and selection of chimeric genes coding for N- or C-terminal His-tagged proteins. These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that were generated by using thermostable polymerases that provide proofreading activity.

RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin ( ori ) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.

Two clearly opposing views exist on the function of alpha-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, in the sexual differentiation of the rodent brain. AFP has been proposed to either prevent the entry of estrogens or to actively transport estrogens into the developing female brain. The availability of Afp mutant mice (Afp(-/-)) now finally allows us to resolve this longstanding controversy concerning the role of AFP in brain sexual differentiation, and thus to determine whether prenatal estrogens contribute to the development of the female brain. Here we show that the brain and behavior of female Afp(-/-) mice were masculinized and defeminized. However, when estrogen production was blocked by embryonic treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione, the feminine phenotype of these mice was rescued. These results clearly demonstrate that prenatal estrogens masculinize and defeminize the brain and that AFP protects the female brain from these effects of estrogens.

α-Fetoprotein (AFP) is a serum protein expressed during fetal life, the expression of which is shut off after birth. The activity of the mouse Afp gene promoter region comprised between −80 and −38 bp is regulated by the thyroid hormone receptor (T3R): negatively in the presence of T3 and positively in the absence of T3. The stimulating effect of unliganded T3R is, unexpectedly, antagonized by cofactors that have histone-acetyl-transferase activity, or by sodium butyrate, which inhibits histone acetylases (HDACs). The unliganded T3R stimulating activity effect is thus associated with protein deacetylation, contrary to the usual situation. In combination with previous results, our observations suggest that T3-mediated down regulation of the Afp promoter is due to T3-induced protein acetylation leading to loss of a nucleosomal structure (required for promoter activity) and chromatin opening.

Using the sequence of the SP1 zinc-finger DNA-binding domain as a probe to screen a mouse EST database, we identified two novel members of the SP/XKLF transcription factor family, KLF13 and KLF14. The mouse Klf13 cDNA (1310 bp in length) contains a single open reading frame of 288 amino acids with a DNA-binding domain closely related to that of the human RFLAT-1 protein and a putative transactivator N-terminal domain rich in proline and alanine residues. The mouse Klf13 gene seems to be the homologue of the human RFLAT1 gene. The mouse Klf14 sequence is homologous to a human genomic sequence from chromosome 17 that is believed to code for a protein with three zinc fingers at the end of its C-terminal domain. Using reverse transcription-polymerase chain reaction, we showed ubiquitous expression of Klf13 and Klf14 in adult mice. A third member of this family was also identified in a human EST database; this sequence was found to be identical to KLF11 (TIEG2), recently identified by Cook et al. (1998, J. Biol. Chem. 273: 25929-25936). The corresponding mouse cDNA was isolated and sequenced. The three genes were localized in the human and the rat: chromosomes 15 (human KLF13), 17q21.3-q22 (human KLF14; HGMW-approved symbol SP6), and 2p25 (human KLF11) and chromosomes 1q31-q32 (rat Klf13), 10q31-q32.1 (rat Klf14) (SP6), and 6q16-q21 (rat Klf11).

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.

The use of PCR-amplified DNA-fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent Aspel sites and a unique HindII site. Cleavage of these vectors with Aspel produce linearized molecules with a single thymidine nucleotide at the 3' ends allowing TA cloning of Taq-amplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZ alpha-CcdB fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15. This bifunctionality allowed us to show that small PCR products (< 1000 bp) that do not disrupt lacZ alpha efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.

RepHI1B is one of the replicons that is specific to IncHI1 multireplicon plasmids. Its general organization resembles that of several replicons that control their copy number by an iteron mechanism. The RepHI1B replicon (2.4 kb) contains: (i) an 882 bp repA gene coding for a 32 kDa replication protein (RepA), sharing significant similarity with the initiator proteins of other replicons belonging to various incompatibility (Inc) groups, including P1 (IncY), Rts1 (IncT), RepFIB (IncFI), and RepHI1A (IncHI1); (ii) two sets of 17 bp DNA repeats (iterons), one upstream and one downstream from repA. By complementation testing, we identified the replication origin (ori) of RepHI1B in a 223 bp locus upstream from repA. By primer extension we mapped two promoters of repA (Pr1 and Pr2) in the ori sequence. We used repA::lacZ transcriptional fusions to study regulation of the repA gene. This analysis showed that repA is transcriptionally autoregulated. Gel mobility shift assays demonstrated that RepA binds specifically to the origin and to iterons overlapping the Pr1 and Pr2 promoters. A G to A transition at nucleotide position 13 of the iteron located in Pr2 (repeat 5) drastically decreases autoregulation of repA by inhibiting binding of RepA.

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

The IncHI1 plasmids are multireplicon plasmids. They contain at least three autoreplicative regions, one of which is closely related to the RepFIA replicon of F. Two other IncHI1-specific replicons, RepHI1A and RepHI1B, have been recently isolated and mapped on the R27 (IncHI1) genome (P. Gabant, P. Newnham, D. Taylor, and M. Couturier, J. Bacteriol. 175, 76977701, 1993). In the present work, the DNA sequence of RepHI1B was determined. It reveals DNA repeats of 17 base pairs located upstream and downstream from a gene coding for a 32 kilodalton protein (RepA) required for replication. Interestingly, RepA presents significant homology with other Rep proteins encoded by plasmids belonging to different incompatibility groups: P1 (IncY), Rts1 (IncT), RepFIB (IncFI) and RepHI1A (IncHI1). All these results provide strong evidence that the RepHI1B replicon of the IncHI1 subgroup belongs to the group of plasmids which control their copy number by an iteron mechanism.