Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

Background The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT) ortholog, and provide a comparison with genes of other vertebrates. Results The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in rayfinned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. Conclusions OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.Radmila Hrdličková, Jiří Nehyba, Shu Ly Lim, Frank Grützner, Henry R Bose J

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin

ORGANIZATION OF AEROBACTIN, HEMOLYSIN, AND ANTIBACTERIAL RESISTANCE GENES IN LACTOSE-NEGATIVE ESCHERICHIA-COLI STRAINS OF SEROTYPE O4 ISOLATED FROM CHILDREN WITH DIARRHEA

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin

Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and the VirF and VirB regulatory cascade

The role in virulence of the Shigella flexneri ospB-phoN2 operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of phoN2, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization

Expression of the virulence plasmid-carried apyrase gene (apy) of enteroinvasive Escherichia coli and Shigella flexneri is under the control of H-NS and VirF and VirB regulatory cascade

The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and enteroinvasive Escherichia coli (EIEC) is induced at 37 degreesC and repressed at 30 degreesC. In this work, we report that the O135: K-:H- EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Deltahns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb-) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb- mutants of HN280 and SFZM53, respectively. While all Crb- apy+ mutants of HN280 failed to produce apyrase, Crb- apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb- mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria

H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome.

We have previously shown that integration of the virulence plasmid pINV into the chromosome of enteroinvasive Escherichia coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59:792-799, 1991). In this work, we have studied the transcription of the virulence regulatory genes virB, virF, and hns (virR) in wild-type enteroinvasive E. coli HN280 and in its pINV-integrated derivative HN280/32. While transcription of virF and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integration does not occur within the virB locus. This indicates that VirF cannot activate virB transcription when pINV is integrated, and this lack of expression accounts for the noninvasive phenotype of HN280/32. Virulence gene expression in strains HN280 and HN280/32, as well as in derivatives harboring a mxiC::lacZ operon fusion either on the autonomously replicating pINV or on the integrated pINV, was studied. The effect of the introduction of plasmids carrying virB (pBNI) or virF (pHW745 and pMYSH6504), and of a delta hns deletion, in the different strains was evaluated by measuring beta-galactosidase activity, virB transcription, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contact-mediated hemolysis, and capacity to invade HeLa cells. The introduction of pBN1 or of the delta hns deletion in pINV-integrated strains induces temperature-regulated expression or temperature-independent expression, respectively, of beta-galactosidase activity and of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Moreover, a wild-type hns gene placed in trans fully reversed the induction of beta-galactosidase activity due to the delta hns deletion. These results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37 degrees C in pINV-integrated strains and that there is also a dose-dependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive temperature of 37 degrees C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-NS to the virB promoter DNA region. At 30 degrees C, the introduction of the high-copy-number plasmid pMYSH6504 (but not of the low-copy-number pHW745) or of the deltahns deletion induces, in strains harboring an autonomously replicating pINV, beta-galactosidase activity, virB transcription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with E. coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in enteroinvasive bacteria, the level of Virf inside the cell controls the temperature-regulated expression of invasion genes