Characteristics of the ErmK Protein of Bacillus halodurans C-125

Abstract

Bacillus halodurans C-125 is an alkaliphilic microorganism that grows best at pH 10 to 10.5. B. halodurans C-125 harbors the erm (erythromycin resistance methylase) gene as well as the mphB (macrolide phosphotransferase) and putative mef (macrolide efflux) genes, which confer resistance to macrolide, lincosamide, and streptogramin B (MLS_B) antibiotics. The Erm protein expressed in B. halodurans C-125 could be classified as ErmK because it shares 66.2% and 61.2% amino acid sequence identity with the closest ErmD and Erm(34), respectively. ErmK can be regarded as a dimethylase, as evidenced by reverse transcriptase analysis and the antibiotic resistance profile exhibited by E. coli expressing ermK. Although ErmK showed one-third or less in vitro methylating activity compared to ErmC', E. coli cells expressing ErmK exhibited comparable resistance to erythromycin and tylosin, and a similar dimethylation proportion of 23S rRNA due to the higher expression rate in a T7 promoter-mediated expression system. The less efficient methylation activity of ErmK might reflect an adaption to mitigate the fitness cost caused by dimethylation through the Erm protein presumably because B. halodurans C-125 has less probability to encounter the antibiotics in its favorable growth conditions and grows retardedly in neutral environments. IMPORTANCE Erm proteins confer MLS_B antibiotic resistance (minimal inhibitory concentration [MIC] value up to 4,096 μg/mL) on microorganisms ranging from antibiotic producers to pathogens, imposing one of the most pressing threats to clinics. Therefore, Erm proteins have long been speculated to be plausible targets for developing inhibitor(s). In our laboratory, it has been noticed that there are variations in enzymatic activity among the Erm proteins, Erm in antibiotic producers being better than that in pathogens. In this study, it has been observed that Erm protein in B. halodurans C-125 extremophile is a novel member of Erm protein and acts more laggardly, compared to that in pathogen. While this sluggishness of Erm protein in extremophile might be evolved to reduce the fitness cost incurred by Erm activity adapting to its environments, this feature could be exploited to develop the more potent and/or efficacious drug to combat formidably problematic antibiotic-resistant pathogens.

Keywords: antibiotic resistance; extromophile; fitness cost; methylases.

FIG 1

Sequence alignment of ErmK and ErmD. ErmK is originated from B. halodurans C-125, and ErmD is from B. licheniformis (42) and one of the closest members to ErmK, the other being Erm(34). When the sequences of two proteins were aligned, 188 positions were recognized to be occupied with the same amino acids so that two proteins exhibited 66.2% amino acid sequence identity which is less than 80%. Therefore, along with the sequence comparison result of Erm(34) and ErmK, 61.2% amino acid sequence identity (Fig. S1), it could be considered to be reasonable and correct that ErmK might be classified as a new member of Erm protein and given a new letter designation of ErmK. The sequence alignment was generated using CLUSTALX2 (40), and the resulting alignment was visualized using ESPript (41).

FIG 2

SDS-PAGE analysis of Erm protein expression (ErmC′ and ErmK) in E. coli and purification (ErmK). (a) Proteins were expressed in the presence of ITPG, inducer (+) or without it (−). To separate the soluble proteins (sup) from the precipitated proteins (ppt, inclusion body fraction), centrifugation (20,000 × g, 30 min) was carried out after disruption of cells by freezing and thawing. Arrowhead indicates ErmC′ (28.907 kDa, calculated) and ErmK (32.336 kDa), respectively. a, the relative amount of each protein was measured using Multi Gauge (ver3.0). The protein amount of ErmC′ expressed in soluble fraction without inducer was set to 1 and the amount of other protein was indicated in the relative amount to it. (b) The soluble fraction of protein (ErmK) was purified on immobilized Ni²⁺affinity column. Lane 1, total cell proteins; lane 2, inclusion body fraction; lane 3, supernatant fraction of lysate; lane 4, affinity runthrough; lane 5, 1 × binding buffer column wash; lane 6, 100 mM imidazole column wash; lane 7, purified soluble ErmK protein (arrowed).

FIG 3

Antibiotic susceptibility assay with E. coli cells expressing ErmC′, ErmN and ErmK and E. coli containing only empty vector. Overnight grown cells was spread on quartered agar plate and circular filter paper containing 1,000 μg of tylosin and erythromycin was placed in the middle of culture. E. coli cells with empty vector exhibit reasonably wide inhibition zone caused by each antibiotic. Presumably tylosin experiences more outer membrane permeability barrier than erythromycin, forming smaller inhibition zone. With monomethylation (ErmN), the inhibition around each antibiotic reduced to form smaller but similar size of inhibition zone. With ErmC′ and ErmK, no inhibition zone could be observed indicating ErmK could be dimethyltransferase.

FIG 4

Autoradiogram of reverse transcripts of in vivo-methylated 23S rRNA in the presence of various Erm proteins. The rRNA was isolated from E. coli cells with or without IPTG induction which harbor plasmid empty pET23b, pHJJ402 (ErmN), pHJJ502 (ErmC′) and pHJJ602 (ErmK). All the 23S rRNAs were analyzed in the same manner by extension with reverse transcriptase from a primer (oligo-7) complementary to nucleotides 2061 to 2078 in E. coli 23S rRNA to assess the degree of methylation. Primer extension should start at A2060 and was designed to terminate at G2057 by the addition of ddCTP if there was no methylation (empty vector) or monomethylation (ErmN) at A2058 (no dimeth). The reverse transcription proceeded in the presence of only dTTP and ddCTP. But dimethyation at A2058 induced primer extension termination at A2059 (M⁶₂2058A). The relative amounts of dimethylated and unmethylated or monomethylated RNA was inferred from the ratio between band intensity at A2059 and band intensity at A2057 plus that at A2059 and shown at the bottom of the autoradiogram (% dimethylation). Analysis with induced strain is indicated by + and that of leaky expression (without induction) is indicated by −. The 23S rRNA template for the dideoxy-sequencing reactions (ATGC) was from a sensitive E. coli strain harboring plasmid pET23b.

FIG 5

72 nt substrate rRNA sequence used for measuring the methyl group transferring activity of ErmK and ErmC′. 72 nt substrate RNA is composed of helix 73 and 74 and from B. subtilis BD170. To facilitate the production of substrate RNA, the ends of helix 73 and 74 were capped with UUCG tetraloop (red) that all the sequences reside on one string of RNA strand. The methylatable adenine is encircled in red.