KpnBI is the prototype of a new family (IE) of bacterial type I restriction-modification system

Abstract

KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5' specific region and the adenine opposite the italicized thymine in the 3' specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new 'type IE' family.

Figure 1

(A) Restriction map of pVC1 that contains modification genes (hsdM and hsdS) of KpnBI. The vector contains one KpnBI site. (B) The 8.2 kb EcoRI fragment and the subclones used for DNA sequencing. Pmod indicates the position of the promoter.

Figure 2

KpnBI HsdS protein sequence deduced from the nucleotide sequence. Several repeats are shown in bold and underlined.

Figure 3

(A) Oligonucleotides used for the confirmation of the KpnBI site and (B) determination of the methylated adenines. KpnBI recognition sequences are shown in bold. All the oligonucleotides contain half of the EcoRV sites (ATCGAT) at each end for blunt-end cloning. Other recognition sites are underlined. Note that the B5 and B6 pair contains G/C differences in the sequence, whereas the rest of the oligonucleotide pairs (A1–A2, B1–B2 and B3–B4) contain A/G differences. Plasmids containing these oligonucleotide sequences are shown in parenthesis.

Figure 4

HindIII digestion of KpnBI modified and unmodified plasmids. Modified plasmids contain a symbol (.m). The various oligonucleotides carried by each plasmid are shown in Figure 3. The arrow points to the 209 bp bands.

Figure 5

Estimation of the conserved and variable regions of the HsdS_KpnBI sequence and its homologs. The five putative HsdS homolog sequences (Table 3B) were aligned using the ClustalW program. Amino acid sequences with more than 50% homology as well as 100% homology are also shown. Amino acid sequences used are from top to bottom: M.jannaschii DSM2661 [originally called Methanococcus jannaschii which contains two HsaS proteins (34)], Shewanella oneidensis MR-1 (35), Methanosarcina mazei Go1 (29), KpnBI (this study) and Cyanobacteria Nostoc sp. PCC7120 (36).