Mónika Szabó

The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish

Early steps of embryo development are directed by maternal gene products and trace levels of zygotic gene activity in vertebrates. A major activation of zygotic transcription occurs together with degradation of maternal mRNAs during the midblastula transition in several vertebrate systems. How these processes are regulated in preparation for the onset of differentiation in the vertebrate embryo is mostly unknown. Here, we studied the function of TATA‐binding protein (TBP) by knock down and DNA microarray analysis of gene expression in early embryo development. We show that a subset of polymerase II‐transcribed genes with ontogenic stage‐dependent regulation requires TBP for their zygotic activation. TBP is also required for limiting the activation of genes during development. We reveal that TBP plays an important role in the degradation of a specific subset of maternal mRNAs during late blastulation/early gastrulation, which involves targets of the miR‐430 pathway. Hence, TBP acts as a specific regulator of the key processes underlying the transition from maternal to zygotic regulation of embryogenesis. These results implicate core promoter recognition as an additional level of differential gene regulation during development.

Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage λ causes transposition primarily into the vicinity of the λ operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.

Analysis of the N‐terminal DNA binding domain of the IS30 transposase

IS30 is the founding member of a large family of widely spread bacterial insertion sequences with closely related transposases. The N‐terminal end of the IS30 transposase had been shown to retain sequence‐specific DNA binding activity and to protect the IS30 terminal inverted repeats. Structural predictions revealed the presence of a helix–helix–turn–helix motif (H–HTH2) which, in the case of IS30, is preceded by an additional helix–turn–helix motif (HTH1). Analysis of deletion and point mutants in this region revealed that both motifs are important for IS30 transposition. IS30 exhibits two types of insertion specificity preferring either a 24 bp palindromic hot‐spot (GOHS) or sequences resembling its ends [left and right terminal inverted repeat (IRL and IRR)]. Results are presented suggesting that the HTH1 region is required for GOHS targeting and interferes with the inverted repeat (IR) targeting. On the other hand, H–HTH2 appears to be required for both. The binding activities of the mutant proteins to the terminal IS30 IRs as measured by gel retardation correlated well with these results. Furthermore, close inspection of the H–HTH2 region revealed significant amino acid identity with a similar predicted secondary structure carried by the transcriptional regulator FixJ of Sinorhizobium meliloti and involved in FixJ binding to its target sequence. This suggests that FixJ and IS30 transposase share similar sequence‐specific DNA binding mechanisms.

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential.

Site-specific recombination by the DDE family member mobile element IS30 transposase

DNA rearrangements carried out by site-specific recombinases and transposases (Tpases) show striking similarities despite the wide spectrum of the catalytic mechanisms involved in the reactions. Here, we show that the bacterial insertion sequence (IS)30 element can act similarly to site-specific systems. We have developed an inversion system using IS30 Tpase and a viable λ phage, where the integration/excision system is replaced with IS30. Both models have been proved to operate analogously to their natural counterpart, confirming that a DDE family Tpase is able to fulfill the functions of site-specific recombinases. This work demonstrates that distinction between transposition and site-specific recombination becomes blurred, because both functions can be fulfilled by the same enzyme, and both types of rearrangements can be achieved by the same catalytic mechanisms.

The master regulator of IncA/C plasmids is recognized by the Salmonella Genomic island SGI1 as a signal for excision and conjugal transfer

The genomic island SGI1 and its variants, the important vehicles of multi-resistance in Salmonella strains, are integrative elements mobilized exclusively by the conjugative IncA/C plasmids. Integration and excision of the island are carried out by the SGI1-encoded site-specific recombinase Int and the recombination directionality factor Xis. Chromosomal integration ensures the stable maintenance and vertical transmission of SGI1, while excision is the initial step of horizontal transfer, followed by conjugation and integration into the recipient. We report here that SGI1 not only exploits the conjugal apparatus of the IncA/C plasmids but also utilizes the regulatory mechanisms of the conjugation system for the exact timing and activation of excision to ensure efficient horizontal transfer. This study demonstrates that the FlhDC-family activator AcaCD, which regulates the conjugation machinery of the IncA/C plasmids, serves as a signal of helper entry through binding to SGI1 xis promoter and activating SGI1 excision. Promoters of int and xis genes have been identified and the binding site of the activator has been located by footprinting and deletion analyses. We prove that expression of xis is activator-dependent while int is constitutively expressed, and this regulatory mechanism is presumably responsible for the efficient transfer and stable maintenance of SGI1.

Characterization of Two Multidrug-Resistant IncA/C Plasmids from the 1960s by Using the MinION Sequencer Device

ABSTRACT Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARIR16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes blaTEM-1D and aphA1b and a mer module, respectively; a truncated Tn5393 copy; and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6-kb ARI inserted into the kfrA gene. ARIIP40a carrying blaTEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARIR16a. Loss of resistance markers in R16a, IP40a, and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. The MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full-length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy-number plasmids and their rearrangement products.

Determination and Analysis of the Putative AcaCD-Responsive Promoters of Salmonella Genomic Island 1

The integrative genomic island SGI1 and its variants confer multidrug resistance in numerous Salmonella enterica serovariants and several Proteus mirabilis and Acinetobacter strains. SGI1 is mobilized by the IncA/C family plasmids. The island exploits not only the conjugation apparatus of the plasmid, but also utilizes the plasmid-encoded master regulator AcaCD to induce the excision and formation of its transfer-competent form, which is a key step in the horizontal transfer of SGI1. Triggering of SGI1 excision occurs via the AcaCD-dependent activation of xis gene expression. AcaCD binds in Pxis to an unusually long recognition sequence. Beside the Pxis promoter, upstream regions of four additional SGI1 genes, S004, S005, S012 and S018, also contain putative AcaCD-binding sites. Furthermore, SGI1 also encodes an AcaCD-related activator, FlhDCSGI1, which has no known function. Here, we have analysed the functionality of the putative AcaCD-dependent promoter regions and proved their activation by either AcaCD or FlhDCSGI1. Moreover, we provide evidence that both activators act on the same binding site in Pxis and that FlhDCSGI1 is able to complement the acaCD deletion of the IncA/C family plasmid R16a. We determined the transcription start sites for the AcaCD-responsive promoters and showed that orf S004 is expressed probably from a different start codon than predicted earlier. Additionally, expression of S003 from promoter PS004 was ruled out. Pxis and the four SGI1 promoters examined here also lack obvious -35 promoter box and their promoter profile is consistent with the class II-type activation pathway. Although the role of the four additionally analysed AcaCD/FlhDCSGI1-controlled genes in transfer and/or maintenance of SGI1 is not yet clear, the conservation of the whole region suggests the existence of some selection for their functionality.

Functional Organization of the Inverted Repeats of IS30

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.

Genome Sequences of Multidrug-Resistant Salmonella enterica subsp. enterica Serovar Infantis Strains from Broiler Chicks in Hungary

ABSTRACT Three strains of Salmonella enterica serovar Infantis isolated from healthy broiler chickens from 2012 to 2013 have been sequenced. Comparison of these and previously published S. Infantis genome sequences of broiler origin in 1996 and 2004 will provide new insight into the genome evolution and recent spread of S. Infantis in poultry.