The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.     

Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN(8-)TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5' truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.     

Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus

Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA. The genome of M. fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene. The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene. The secondary structures predicted for the M. fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria. A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs.     

Biosynthetic Pathway of Cephabacins in Lysobacter lactamgenus: Molecular and Biochemical Characterization of the Upstream Region of the Gene Clusters for Engineering of Novel Antibiotics

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.     

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.     

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Molecular analysis of a complex locus from Haemophilus influenzae invoked in phase-variable lipopolysaccharide biosynthesis

A chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. lic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. ORF2 encodes the UDP-galactose-4-epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. ORF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3' end of ORF1, all of galE, and the 5' end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.     

The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4

A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.     

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.