Discovery and distribution of super-integrons among pseudomonads.

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of 'super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.

Comparative genomics of the restriction-modification systems in Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64-1.67 Mb. More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.

The Ketel gene encodes a Drosophila homologue of importin-beta.

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.

Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome.

Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently. About two dozen potential restriction-modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes. Here we describe a functional analysis of the 16 putative Type II R-M systems in the H. pylori J99 genome. To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements. To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine. We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities. Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial 'immune' system, by alternatively turning on/off a subset of numerous R-M systems.

Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.

VISA: Visual Sequence Analysis for the comparison of multiple amino acid sequences.

VISA (VIsual Sequence Analysis) is a software package that displays global similarities within a set of related protein sequences. The program identifies amino acid patterns that are common to many members of the set of sequences and displays them as a series of histograms. Individual peaks on the display can be assigned a color and analogous peaks in the other sequences are then automatically marked in the same color. This can be repeated for each significant peak and leads to a display in which major matching segments of multiple amino acid sequences appear as dominant peaks of the histograms with matching colors. These peaks usually correspond to the conserved sequence motifs that are characteristic of particular proteins. An extensive set of software tools is included to help the localization, visualization and analysis of the global similarities displayed. VISA provides a graphic overview of the sequence similarity that can help to understand the architecture of the protein family and can be helpful while designing experiments to probe function.

The DNA (cytosine-5) methyltransferases.

The m5C-MTases form a closely-knit family of enzymes in which common amino acid sequence motifs almost certainly translate into common structural and functional elements. These common elements are located predominantly in a single structural domain that performs the chemistry of the reaction. Sequence-specific DNA recognition is accomplished by a separate domain that contains recognition elements not seen in other structures. This, combined with the novel and unexpected mechanistic feature of trapping a base out of the DNA helix, makes the m5C-MTases an intriguing class of enzymes for further study. The reaction pathway has suddenly become more complicated because of the base-flipping and much remains to be learned about the DNA recognition elements in the family members for which structural information is not yet available.

Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine.

The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.

Finding errors in DNA sequences.

An algorithm is described that can detect certain errors within coding regions of DNA sequences. The algorithm is based on the idea that an insertion or deletion error within a coding sequence would interrupt the reading frame and cause the correct translation of a DNA sequence to require one or more frameshifts. If the coding sequence shows similarity to a known protein sequence then such errors can be detected by comparing the conceptual translations of DNA sequences in all six reading frames with every sequence in a protein sequence data base. We have incorporated these ideas into a computer program, called DETECT, that can serve as an aid to the experimentalist who is determining new DNA sequences so that obvious errors may be located and corrected. The program has been tested using raw experimental data and against sequences from the European Molecular Biology Laboratory data base, annotated as containing frameshifts. We have also tested it using unidentified open reading frames that flank known, annotated genes in the GenBank data base. Many potential errors are apparent and in some cases functions can be suggested for the "corrected" versions of these reading frames leading to the identification of new genes. As more sequences are determined the power of this method will increase substantially.

Predictive motifs derived from cytosine methyltransferases.

Thirteen bacterial DNA methyltransferases that catalyze the formation of 5-methylcytosine within specific DNA sequences possess related structures. Similar building blocks (motifs), containing invariant positions, can be found in the same order in all thirteen sequences. Five of these blocks are highly conserved while a further five contain weaker similarities. One block, which has the most invariant residues, contains the proline-cysteine dipeptide of the proposed catalytic site. A region in the second half of each sequence is unusually variable both in length and sequence composition. Those methyltransferases that exhibit significant homology in this region share common specificity in DNA recognition. The five highly conserved motifs can be used to discriminate the known 5-methylcytosine forming methyltransferases from all other methyltransferases of known sequence, and from all other identified proteins in the PIR, GenBank and EMBL databases. These five motifs occur in a mammalian methyltransferase responsible for the formation of 5-methylcytosine within CG dinucleotides. By searching the unidentified open reading frames present in the GenBank and EMBL databases, two potential 5-methylcytosine forming methyltransferases have been found.

Sequence motifs specific for cytosine methyltransferases.

Using a new alignment method, the sequences of 13 m5C methyltransferases (MTases) have been examined. Five extremely well-conserved blocks of sequence have been detected and have been used as fixed points for the alignment of the 13 sequences. Following this initial alignment, five further blocks of similarity have been identified to give a total of ten recognizable blocks of sequence homology that are all arranged in a common order. The structures of these MTases consist of a variable-length N-terminal arm followed by eight well-conserved blocks each separated by small variable-length regions. A large variable-length segment of 90 to 270 amino acids (aa) then follows. After this are two blocks, and a variable-length C-terminal segment completes the sequence. Within the final alignment, 20 aa in the protein sequences, and 86 nucleotides in the nucleotide sequences are invariant. The strongest conservation is found in proximity to a suspected functional site that contains the dipeptide proline-cysteine. Consensus patterns can be defined for the five best conserved blocks and, when used as search motifs, are able to clearly distinguish between the m5C MTases and all other identified proteins in the PIR database. This suggests they may be of use in identifying putative MTases among protein sequences of unknown function.

Structure of the gene coding for the sequence-specific DNA-methyltransferase of the B. subtilis phage SPR.

The nucleotide sequence of the gene coding for the 5'-GGCC and 5'-CCGG specific DNA methyltransferase of the Bacillus subtilis phage SPR was determined by the Maxam-Gilbert procedure. Transcriptional and translational signals of the sequence were assigned with the help of S1 mapping and translation in E. coli minicells. The gene codes for a 49 kd polypeptide. The amino acid sequence of the SPR methylase shows regions of homology with the sequence of the 5'-GGCC-specific BspRI modification methylase.

Nucleotide sequence of Rhizobium meliloti nodulation genes.

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.

Structure of the Bacillus sphaericus R modification methylase gene.

A 2.5 X 10(3) base-pair segment of Bacillus sphaericus R DNA cloned in Escherichia coli has previously been shown to carry the functional BspRI modification methylase gene. The approximate location of the gene on this DNA segment and its direction of transcription were established by subcloning experiments. The nucleotide sequence of the relevant region was determined by the Maxam-Gilbert procedure. An open reading frame that can code for a 424 amino acid protein was found. The calculated molecular weight (48,264) of this protein is in fair agreement with previous estimates (50,000 to 52,000). The synthesis of this protein was demonstrated in E. coli minicells. The initiation point of transcription by E. coli RNA polymerase was localized by in vitro transcription experiments. The open reading frame starts 29 base-pairs downstream from the transcription initiation site and it is preceded by a sequence showing extensive Shine-Dalgarno complementarity. Subcloning experiments and translation in minicells suggest that after removal of this translational initiation site, a secondary start site 29 amino acids downstream can also start translation in E. coli, and this shorter protein retains the methylase activity. The overall base composition of the gene and the codon usage indicate a strong preference for A.T base-pairs.

Sequence homology between potato spindle tuber viroid and U3B snRNA.

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.