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PubMed2003

Variation between pathogenic serovars within Salmonella pathogenicity islands.

Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S. enterica. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E. coli retron phage PhiR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into S. enterica and that in some cases the same event has occurred in multiple lineages of S. enterica. Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic Salmonella serovars.

Amino Acid Sequence↗
PubMed1993

Studies of bacteriophage P2 DNA replication. The DNA sequence of the cis-acting gene A and ori region and construction of a P2 mini-chromosome.

A self-replicating plasmid was constructed from the 76.7 to 91.6% region of bacteriophage P2, which contains the P2 origin or replication (ori) and the genetically defined replication genes B and A. The sequence of the 76.7 to 80.2% has been determined previously, and the sequence of the 80.2 to 91.6% region is now reported. The sequenced region contained gene A, which predicts a 761 amino acid residue polypeptide known to induce a single-strand cut at or near ori, and ori, which has been located by electron microscopy to about 89% from the left end of the phage genome. Analysis of plasmid-encoded proteins in minicells indicated that the A gene product was about 78 kilodaltons. Five previously unknown open reading frames (orf-80, orf-81, orf-82, orf-83 and orf-91) were discovered. They have been cloned and their respective products were identified. The products of orf-80, orf-82 and gene A were found to be lethal to the host when overexpressed. The predicted amino acid sequences of the orf-82 and orf-83 gene products were similar to two early gene products of phage 186; the orf-91 product resembled the hypothetical protein of orfd of retron Ec67. Similarities between the products of P2 gene A, 186 gene A and orf2 and orf3 of Ec67 were also found. A P2 mini-chromosome has been constructed that contains only the P2 A gene and the beta-lactamase gene as a marker.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of host mutants defective in msDNA synthesis: role of ribonuclease H in msDNA synthesis.

In some Escherichia coli strains, a single-stranded DNA (msDNA) covalently linked to RNA is produced by a reverse transcriptase (RT) encoded by a genetic element called a retron. We have looked for host genes involved in msDNA synthesis. From screening of 10,000 mutants generated by Tn5 insertions, we obtained 3 chromosomal mutants defective msDNA synthesis. Analysis showed that all 3 mutants were affected in the gene for ribonuclease H (rnh). In rnh mutants, the reverse transcription products were heterogeneous with several sizes, smaller and bigger than the wild-type msDNA, showing that the reverse transcription is inhibited by the lack of RNase H activity. It also suggests that the RT recognizes the structure of msDNA for the termination of reverse transcription.

Chromosome Mapping↗
PubMed1995

Structure, function, and evolution of bacterial reverse transcriptase.

The discovery of retroelements in the prokaryotes raises intriguing questions concerning their roles in bacteria and the origin and evolution of reverse transcriptases. We first discuss a possible structure of bacterial reverse transcriptases on the basis of the known three-dimensional structure of HIV-1 reverse transcriptase, and how such a putative three-dimensional structure is able to recognize a single primer-template RNA molecule to initiate DNA chain elongation from the 2'-OH group of an internal G residue. This reaction leads to the production of a unique RNA-DNA complex called msDNA (multicopy single-stranded DNA) in which a single-stranded DNA branches out from an RNA molecule via a 2',5'-phosphodiester linkage. Second, the mobility of the bacterial retroelements called retrons, responsible for the production of msDNA, are discussed and compared with the mobility of group I and group II introns. Third, the original and evolution of bacterial reverse transcriptases are discussed in light of the question of whether the bacterial reverse transcriptases are older than eukaryotic reverse transcriptases.

Amino Acid Sequence↗
PubMed1995

Retroelements, reverse transcriptase and evolution.

Retroelements are genetic elements that can exist as DNA or RNA or DNA/RNA duplexes. Although retroviruses are the best known retroelements, there are many other types, including close relatives of retroviruses like LTR retrotransposons, more distant relatives like non-LTR retrotransposons, caulimoviruses and hepadnaviruses and elements with virtually no similarity, like retrons. Virtually all retroelements are 'selfish DNAs' with no involvement with the normal development or maintenance of their host cells, the only known exception being telomereres/telomerases which maintain the ends of chromosomes. Virtually all retroelements use tRNA, or RNA with strong secondary structure, to initiate their reverse transcription. The coincidence between the use of tRNA, a molecule central to the conversion of RNA to protein, with reverse transcriptase, an enzyme which is crucial for the conversion of RNA to DNA is striking, because RNA probably preceded DNA and protein in evolution. It seems plausible that retroelements were present at the genesis of living systems.

Base Sequence↗
PubMed2006

Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin analogues.

INTRODUCTION: Bombesin, a neuropeptide with potential for breast and prostate tumor targeting, is rapidly metabolized in vivo, and as a result, uptake in tumor xenografts in mice is poor. An improvement can be expected from the introduction of nonnatural amino acids and spacers. Leu13 was replaced by cyclohexylalanine and Met14 by norleucine. Two spacers, -betaAla-betaAla- and 3,6-dioxa-8-aminooctanoic acid, were inserted between the receptor-binding amino acid sequence (7-14) of bombesin (BBS) and the retroN(alpha)-carboxymethyl histidine chelator used for labeling with the [99mTc](CO)3 core and the rhenium (Re) congener. METHODS: The biological characterization of the new compounds was performed both in vitro on prostate carcinoma PC-3 cells (binding affinity, internalization/externalization) and in vivo (biodistribution in nude mice with tumor xenografts). The stability was also investigated in human plasma. The Re analogues were prepared for chemical characterization. RESULTS: The nonnatural amino acids led to markedly slower degradation in human plasma and PC-3 cell cultures. The receptor affinity of the new technetium 99m ([99mTc])-labeled BBS analogues was similar to the unmodified compound with Kd<1 nM. Uptake in the pancreas and in PC-3 tumor xenografts in nude mice was blocked by unlabeled BBS. The best target-to-nontarget uptake ratio was clearly due to the presence of the more polar spacer, -betaAla-betaAla-. CONCLUSIONS: The different spacers did not have a significant effect on stability or receptor affinity but had a clear influence on the uptake in healthy organs and tumors. Uptake in the kidneys was lower than in the liver, which is likely to be due to the lipophilicity of the compounds. A specific, high uptake was also observed in the gastrin-releasing peptide receptor-rich pancreas. Thus, with the introduction of spacers the in vivo properties of the compounds can be improved while leaving the affinity unaffected.

Adenocarcinoma↗
PubMed1998

Unusual insertion element polymorphisms in the promoter and terminator regions of the mucAB-like genes of R471a and R446b.

We have previously identified umu-complementing genes on two incL/M plasmids, R471a and R446b (C. Ho et al., J. Bacteriol., 175 (1993) 5411-5419). Molecular analysis of these genes revealed that they are more structurally and functionally related to mucAB from the incN plasmid pKM101 than to other members of the previously identified Umu-like family. As a consequence, we have termed these new homologs mucAB(R471a) and mucAB(R446b) respectively. Interestingly, while the location of the mucAB-like genes is essentially the same in both R471a and R446b, the regions immediately flanking the mucAB-like genes are highly polymorphic. For example, 5' to mucAB(R471a) we found an insert that appears to be a novel retroelement encoding a putative reverse transcriptase (RT). This RT is related to the reverse transcriptases encoded by group II introns but is embedded in a retron-like context. Immediately 3' to the mucAB(R471a) locus is a putative insertion element of a sparsely-dispersed class not previously reported from enteric bacteria. Both the RT and insertion element are absent in R446b. These observations suggest that the mucAB-like genes from R471a and R446b are located within regions of the R-plasmids that perhaps were once (or still are) mobile genetic elements. Such observations might help explain the distribution of umu-like genes on R-plasmids and bacterial chromosomes.

Amino Acid Sequence↗
PubMed1998

Molecular evolution of viruses--Past and Present, Part 2--An introduction.

The evolution of viruses is reviewed within the perspective of the concepts on the evolution of the lipid membrane bound vesicular structures in the prebiotic soup through the ideas on evolution of cells during the RNA World and the transition into the DNA World. The ancient Archeae bacteria and their retrons that carry the bacterial reverse transcriptase gene and their unique protein splicing capability provide an indication of the evolutionary path for retroviruses and, independently, for RNA and DNA viruses of the prokaryotic Archeae bacteria and the eukaryotic yeast and fungi.

Archaea↗
PubMed2026

Pervasive phosphorylation by phage T7 kinase disarms bacterial defences.

Bacteria and bacteriophages are in a constant arms race to develop defence and anti-defence systems, respectively. Currently known phage-encoded anti-defence systems are specific to the activity of the targeted bacterial defence system. Here we identify a mechanism by which the T7 bacteriophage broadly counteracts bacterial defences using protein phosphorylation. Its kinase (T7K), which has been reported to redirect the function of a few host proteins1-5, is actually a hyperpromiscuous dual-specificity kinase that phosphorylates nearly all host and phage proteins during infection. The scale of phosphorylation vastly exceeds known phosphosites in Escherichia coli, has no sequence motif specificity and results in a higher proteome-wide phosphorylation density than mammalian cells with around 500 kinases. Stoichiometry analysis of phosphorylation sites revealed strong bias in T7K activity towards nucleic-acid-binding substrates mediated by its C-terminal DNA-binding domain. This highly stoichiometric phosphorylation enables the deactivation of DNA-targeting or DNA-containing bacterial defence systems. We provide mechanistic insights into how T7K weakens DNA-containing Retron-Eco9 through specific phosphorylation events, with single phosphomimetic mutations in key sites of the toxin abolishing defence. Moreover, by screening a large collection of E. coli strains, we provide evidence of broad anti-defence abilities of T7K in nature, as counteracted strains contain diverse bacterial defence systems. T7K homologues are found almost exclusively in phages, with hyperpromiscuous kinase activity probably being enabled by a divergent DFG-like motif in the catalytic centre.

Journal Article↗
PubMed1995

The formation of the 2',5'-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system.

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.

Base Sequence↗
PubMed1995

Gene regulation by antisense DNA produced in vivo.

Antisense technology has been widely used for regulating gene expression. Single-stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single-stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein.

Bacterial Outer Membrane Proteins↗
PubMed1991

Evolution of retroposons by acquisition or deletion of retrovirus-like genes.

The retroid family consists of all genetic elements that encode a potential reverse transcriptase (RT). Members of this family include a diversity of eukaryotic genetic elements (viruses, transposable elements, organelle introns, and plasmids) and the retrons of prokaryotes. Some retroid elements have, in addition to the RT gene, other genes in common with the retroviruses. On the basis of RT sequence similarity, the retroposon group is defined as the eukaryotic long interspersed nuclear elements, the transposable elements of (1) Drosophila melanogaster (I and F factors), (2) Trypanosoma brucei (ingi element), (3) Zea mays (Cin4), (4) Bombyx mori (R2Bm), and members of the group II introns and plasmids of yeast mitochondria. The data presented here elucidate the extent of the relationships between the retroposons and other retroid-family members. Protein-sequence alignment data demonstrate that subsets of the retroposons contain different assortments of retroviral-like genes. Sequence similarities can be detected between the capsid, protease, ribonuclease H, and integrase proteins of retroviruses and several retroposon sequences. The relationships among the retroposon capsid-like sequences are congruent with the RT sequence phylogeny. In contrast, the similarity between ribonuclease H sequences varies in different subbranches of the retroposon lineage. These data suggest that xenologous recombination (i.e., the replacement of a homologous resident gene by a homologous foreign gene) and/or independent gene assortment have played a role in the evolution of the retroposons.

Amino Acid Sequence↗
PubMed1992

An SOS-inducible defective retronphage (phi R86) in Escherichia coli strain B.

In Escherichia coli, RecA protein regulates the DNA damage-inducible survival-enhancing SOS response. Mutant allele recA730, which causes constitutive SOS expression, is lethal at high temperatures in B/r, a derivative of wild-type B, but not in K-12 or in certain B/r--K-12 hybrids. We present evidence that killing is due to SOS induction of a defective retronphage, phi R86, which is integrated into the B/r chromosome at 19 min, but is absent in K-12. phi R86 contains retron EC-86 which encodes reverse transcriptase and a small multicopy DNA-RNA complex, msDNA-RNA. Induction of phi R86 in recA730 B/r strains results in inhibition of host DNA replication before cell death. A retronphage 'killer' gene, ORF336, when overexpressed from a plasmid, causes similar effects without SOS induction. phi R86 is not detectably u.v.-inducible in recA+ strains.

Coliphages↗
PubMed1996

Enhancement of frame-shift mutation by the overproduction of msDNA in Escherichia coli.

A minor population of wild Escherichia coli strains contain retroelements called retrons, which produce a peculiar satellite DNA, multicopy single-stranded DNA (msDNA). It has been reported that mismatched base pairs in the secondary structure formed in msDNA are mutagenic in E. coli[Maas et al.(1994) Mol.Microbiol. 14,437-441; Maas et al. (1996) Mol. Microbiol, 19, 505-509]. We reexamined this proposal by converting mismatched base pairs to matched base pairs using a single msDNA species, msDNA-Ec86, or by deleting mismatched regions using msDNA-Ec73. We also examined the effect of reverse transcriptases (RT) without msDNA production on mutagenesis. All the constructs are under the lpp/lac promoter-operator control so that their mutagenic effects can be tested in the absence and the presence of a lac inducer. It was found that when the production of msDNA-Ec86 or Ec73 was induced, reversion frequencies from Lac- to Lac+ significantly increased in the case of a Lac- mutation caused by a frame-shift mutation, but much less by a substitution mutation. The removal of mismatched base pairs eliminated the high mutation frequencies, and the inducible expression of RT alone was not mutagenic. These results are consistent with the hypothesis of Maas and his associates that mismatched base pairs in msDNA sequester a cellular mismatch repair system, resulting in the increase of frame-shift mutations.

Base Composition↗
PubMed1996

Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells.

The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.

Adenosine Triphosphatases↗
PubMed2004

Micromanager.

George Latour considers himself a good leader. As CEO of Retronics, George has a mandate to grow revenues with an eye toward taking the software-engineering firm public by 2006. At the behest of the chairman of the board, he has hired a new marketing director, Shelley Stern--"a thoroughbred" who, the chairman insists, just needs a little training in the business. George does his best to bring his new hire up to speed. He has Shelley sit in on developers' meetings, has her accompany the sales force on client calls, and even has the CFO explain the company's cash flow situation to her. He also takes pains to help her correctly position marketing and press materials. But Shelley never seems to really take the bit. In fact, Shelley considers George's hands-on management style oppressive, and she's dreadfully unhappy. What George sees as efforts to bring her up to speed, like making her go on those sales calls when she has other work to do, she views as signs that he doesn't trust her judgment. What's more, Shelley is spread too thin. Yet when she asks for help--if not additional staff, at least an outside contractor--George asks for a list of everything she's working on and tells her he'll help her prioritize. In this fictional case, a he-said, she-said debate erupts over competing management styles. Four commentators--Jim Goodnight, the CEO of SAS Institute; Mark Goulston, a psychiatrist and the senior vice president at Sherwood Partners; J. Michael Lawrie, the CEO of Siebel Systems; and Craig Chappelow, the senior manager of assessment and development resources at the Center for Creative Leadership--offer their perspectives on the problem and how to solve it.

Administrative Personnel↗
PubMed2004

A proteomic approach to study msDNA function in Escherichia coli.

Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

DNA, Bacterial↗