Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli

A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene.     

Deoxyribonucleic acid homologies among strains of Bacteroides melaninogenicus and related species

Saccharolytic, black-pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus ; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius , ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled 'Bact. melaninogenicus subsp. levii' were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus , were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis , and also from strains of related non-pigmented Bacteroides species.     

Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp.

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.     

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.     

Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase

We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.     

Nucleotide sequence of the Thiobacillus ferrooxidans chromosomal gene encoding mercuric reductase

The nucleotide sequence of the Thiobacillus ferrooxidans chromosomal mercuric-reductase-encoding gene (merA) has been determined. The merA gene contains 1635 bp, and shares 78.2% and 76.6% sequence homology with the transposon, Tn501, and plasmid R100 merA genes, respectively. From the sequence, a 545-amino acid (aa) polypeptide was deduced, and comparison with those of Tn501 and R100 revealed 80.6% and 80.0% homology, respectively, at the aa sequence level. Divergence among the three merA aa sequences was clustered within a specific region (aa positions 41-87). By analysis of codon usage frequency, it is speculated that the T. ferrooxidans merA gene originated from Tn501, R100, or a common ancestral gene, but not from T. ferrooxidans itself.     

Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp.

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.     

Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase.

Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.     

Presence of chromosomal elements resembling the composite structure Tn3701 in streptococci.

Tn3701, carried by Streptococcus pyogenes A454, is the first chromosomal composite element to be described; it contains in its central region Tn3703, a transposon similar to Tn916. A comparison by DNA-DNA hybridization of Tn3701 with omega(cat-tet) and Tn3951, carried by Streptococcus pneumoniae BM6001 and by Streptococcus agalactiae B109, respectively, revealed that the two latter structures are also Tn3701-like composite elements. The DNAs of 27 other antibiotic-resistant group A, B, C, and G streptococci and of S. pneumoniae BM4200 showed sequence homologies to Tn3701 (14 strains, including BM4200), to the regions of Tn3701 outside of Tn3703 (5 strains), and to Tn916 alone (8 strains). The DNAs of five strains did not detectably hybridize with any probe. The tetM gene was identified in most chromosomal genetic elements coding for tetracycline-minocycline resistance. Since Tn3701-like elements are widely disseminated among antibiotic-resistant streptococci (47% of the 34 strains studied), we propose that Tn3701 be considered the prototype of chromosomal composite elements.     

Analysis of chromosomal insertion sites for Bacteroides Tn4555 and the role of TnpA

Tn4555, a mobilizable transposon carrying cefoxitin resistance, is directed to a preferred target site in the Bacteroides fragilis chromosome by a transposon-encoded targeting protein TnpA. In an effort to characterize target site selection for Tn4555, the existence of preferred target sites in other species of Bacteroides and in Escherichia coli was examined. For these analyses a Tn4555 mini element, pFD660, was transferred from E. coli donors to Bacteroides thetaiotaomicron or Bacteroides ovatus recipients and the resulting sites of insertion analyzed. A similar construct, pFD794 was used to determine insertion sites in E. coli, and preferred sites were found in all bacteria tested. Also the ability of TnpA to bind to various targets was examined in mobility shift assays. Although TnpA bound to all tested sequences, it displayed higher affinity for the target sites. The binding characteristics of TnpA and the lack of significant base sequence homology between targets suggested that secondary structure of the sites was important for TnpA binding. Circular permutation tests supported the idea that TnpA targets bent DNA.     

N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci

The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.     

Characterization of the 13-kilobase ermF region of the Bacteroides conjugative transposon CTnDOT

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351 element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2, an open reading frame located immediately downstream of the integrase (int) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the Bacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverse Bacteroides population.     

Nucleotide Sequence Determination and Genetic Analysis of theBacteroidesPlasmid, pBI143

The nucleotide sequence and genetic organization of theBacteroidesplasmid pBI143 were determined. The plasmid was 2747 base pairs (bp) and had a G+C content of 41% (GenBank Accession No. U30316). There were two open reading frames greater than 50 codons and these were designatedmobAandrepA.A 56-bp inverted repeat divided pBI143 into modules withrepAandmobAin separate regions. There was a marked difference in the G+C content and codon usage for the two regions;repAhad 33% G+C andmobAwas 44% G+C. MobA had homology to otherBacteroidesmobilization proteins and RepA shared homology to a replication protein fromZymomonas mobilisplasmid pZM2. These two putative replication proteins formed a subgroup of the rolling-circle replication proteins belonging to the pSN2 family of gram-positive plasmids. Consistent with this finding, single-stranded pBI143 DNA was detected in plasmid containingBacteroides fragiliscultures. Availability of the pBI143 sequence allowed the elucidation of the complete nucleotide sequence for pFD288 an 8.9-kbBacteroidesshuttle vector (GenBank Accession No. U30830).     

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.     

Capnocytophaga: New genus of gram-negative gliding bacteria. IV. DNA base composition and sequence homology

Isolates of Gram-negative fermentative gliding bacteria which are prominantly cultivated from the subgingival sulcus in association with periodontal lesions have been the subject of a collaborative taxonomic study. Thirty-five oral strains, isolated from various states of periodontal health and disease, were examined for DNA base composition and patterns of DNA sequence homology. The phentotypically similar organism, Bacteroides ochraceus ATCC 27872, as well as two representatives of gliding bacteria in the family Cytophagaceae, Myxococcus fulvus and Sporocytophaga myxococcoides, were included in these comparisons. Mol-percent guanine and cytosine (% G+C) was determined by thermal denaturation. Relatedness was also assessed by interspecific reassociation of DNA measured by the use of a single-strand specific S1 endonuclease. DNA purified from oral gliders, B. ochraceus ATCC27872 and S. myxococcoides contained 33–41% G+C as compared with 67% in DNA from M. fulvus. Three homology groups (designated as 25, 4 and 27) were delineated by DNA homology. Homology at the 77% level was demonstrated between B. ochraceus ATCC 27872 and the oral reference strain 25. Homology group 4 comprised four strains, all of which were isolated from cases of rapidly advancing periodontal disease. The relatively high degree of genetic divergence, observed as intergroup homology levels of less than 25%, supports the naming of three species of Capnocytophaga, C. ochracea, C. sputigena and C. gingivalis by Leadbetter et al. (1979) corresponding to DNA homology groups 25, 4 and 27, respectively.     

Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation

Tn5520 is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistant Bacteroides fragilis clinical isolate. When a conjugation apparatus is provided in trans, Tn5520 is mobilized (transferred) efficiently within, and from, both Bacteroides spp. and Escherichia coli. Only two genes are present on Tn5520; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn5520 mobility. The focus of this study was to identify the Tn5520 origin of conjugative transfer (oriT) and to study BmpH-oriT binding. We delimited the functional Tn5520 oriT to a 71 bp sequence upstream of the bmpH gene. A plasmid vector harbouring this minimal 71 bp oriT was mobilized at the same frequency as that of intact Tn5520. The minimal oriT contains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5'-GCTAC-3') was also identified within the minimal oriT; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn5520 oriT fragment in electrophoretic mobility shift assays. We also identified non-nick site sequences within the minimal oriT that were essential for mobilization. We hypothesize that transposon-based single Mob protein systems may contribute to efficient gene dissemination from Bacteroides spp., because fewer DNA processing proteins are required for relaxosome formation.