Isolation of An Erythromycin-Resistant (Putative) Gene From Bacillus Halodurans CM1

Abstract

Bacillus halodurans CM1 is one of the bacterial strains from BPPT Culture Collection which had been isolated from sediments in the Cimanggu hot spring, West Java, previously. The whole genomic DNA of this strain has not yet been deeply studied despite its potential as host to produce several kinds of useful enzymes, such as xylanase, lipase, and protease. Besides the industrial enzymes, potential genes in B. halodurans CM1, for example antibiotic-resistant genes, could be engineered later for further purposes. Earlier experiments showed that CM1 strain has the ability to survive in the media with erythromycin, suggesting there might be genes responsible for this erythromycin resistance. This study aimed to isolate erythromycin-resistant gene from B. halodurans CM1 using a PCR approach. The primers used for PCR were designed based on the GenBank database of whole genome Bacillus halodurans C-125. The PCR product was then ligated into a pGEM-T easy cloning vector, and the recombinant vector then transformed into Escherichia coli DH5a. The DNA fragment with the size of 864 bp was obtained and it encoded 288 deduced amino acids. DNA sequence analysis showed that the gene had 99% similarity with gene encoding erythromycin resistance from B. halodurans C-125 (GenBank No access: BA000004.3, ErmK). There are 3 nucleotides in the ErmK sequence of B. halodurans CM1 that are different from that of another well-known strain B. halodurans C-125. This dissimilar pattern of sequence encodes Leu instead of Ser in 155th orders of CM1 amino acid sequence.