Two different restriction-modification systems for degrading exogenous DNA in Paenibacillus polymyxa

Shen, Minjia; Chen, Ziyan; Mao, Xudan; Wang, Lin; Liang, Jingyi; Huo, Qingyuan; Yin, Xiaoyu; Qiu, Juanping; Sun, Dongchang

doi:10.1016/j.bbrc.2018.09.016

Cited by 4 publications

(7 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To express the polymyxin-inactivating enzyme in B. subtilis WB800, the E. coli-B. subtilis shuttle vector pWBUC01 was employed (27). The recombinant plasmid was constructed using the ClonExpress II one-step cloning kit (Vazyme, Nanjing, China) ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…To confirm whether the Apr protein of B. licheniformis DC-1 is indeed a polymyxin-inactivating enzyme, the apr gene was expressed in the heterologous host B. subtilis WB800, which cannot degrade polymyxins, as mentioned above. pWBUC01, a shuttle plasmid for E. coli and B. subtilis, was employed to express exogenous genes under the control of P43, a strong constitutive promoter in B. subtilis (27). When we used this plasmid to clone B. licheniformis apr, we failed to obtain any B. subtilis transformants.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Inactivation of Polymyxin by Hydrolytic Mechanism

Yin

Wang

Cheng

et al. 2019

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Polymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxininactivating enzyme from Bacillus licheniformis strain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other between L-Thr and L-␣-␥-diaminobutyric acid (L-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, including Bacillus and Paenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis. KEYWORDS Bacillus licheniformis, alkaline protease, polymyxin resistance, polymyxininactivating enzyme, polymyxins L-␣-␥-diaminobutyric acid (L-Dab) residues (Fig. 1). Due to the higher affinity between L-Dab and lipid A, polymyxins displace divalent cations and bind to lipid A molecules, resulting in destabilization of the outer membrane. The hydrophobic domains of polymyxins (including the fatty acyl chain and D-Phe/D-Leu-L-Leu) insert between the acyl chains of lipid A and then enter the periplasm via a self-promoted uptake mechanism. Subsequently, polymyxins penetrate into the phospholipid bilayer of the RESULTSPolymyxins can be inactivated by Bacillus species. Studies from several decades ago indicated that colistin-inactivating enzymes are probably produced and secreted by the producer of colistin, P. polymyxa (20)(21)(22). To identify the colistin-inactivating enzyme, the cylinder-plate diffusion method was performed to screen colistin-degrading bacteria from our laboratory stocks of Bacillus strains. Escherichiacoli strain DH5␣ was used as a colistin-sensitive indicator strain. The addition of colistin (10,000 U/ml) resulted in an FIG 1 Chemical structure and degradation pathway of colistin. The amino acid positions are numbered in accordance with the discussion in the text. The amino acid at position 6 is D-Phe in polymyxin B. Both colistin and PMB are mixtures of structurally related compounds. Fatty acid, 6-methyloctanoic acid for colistin A and PMB 1 and 6-methylheptanoic acid for colistin B and PMB 2

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Inactivation of Polymyxin by Hydrolytic Mechanism

Yin

Wang

Cheng

et al. 2019

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…In contrast, P. polymyxa ATCC 842 was phenotypically and genetically stable from one generation to another, making it ideal as a model strain of Paenibacillus . Our previous work established a method for transfer and expression of exogenous genes in P. polymyxa ATCC 842 ( Shen et al, 2018 ). In this study, we have successfully inactivated its chromosomal genes ( Figure 3 ).…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid transformation of P. polymyxa was performed by using our previously established method ( Shen et al, 2018 ). A fresh single colony of P. polymyxa was inoculated into LB medium and incubated at 37°C overnight.…”

Section: Methodsmentioning

confidence: 99%

“…It is also an antibiotic producer which biosynthesizes the last-resort antibacterial drug polymyxin (Choi et al, 2009;Shaheen et al, 2011;Galea et al, 2017) and antifungal drug fusaricidin (Li and Jensen, 2008;Yang et al, 2018). Our previous work revealed the presence of RM systems in P. polymyxa (Shen et al, 2018). However, molecular basis of RM systems and their DNA methylation motifs have yet to be characterized.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Type I Restriction Modification System Influences Genomic Evolution Driven by Horizontal Gene Transfer in Paenibacillus polymyxa

et al. 2021

Self Cite

View full text Add to dashboard Cite

Considered a “Generally Recognized As Safe” (GRAS) bacterium, the plant growth–promoting rhizobacterium Paenibacillus polymyxa has been widely applied in agriculture and animal husbandry. It also produces valuable compounds that are used in medicine and industry. Our previous work showed the presence of restriction modification (RM) system in P. polymyxa ATCC 842. Here, we further analyzed its genome and methylome by using SMRT sequencing, which revealed the presence of a larger number of genes, as well as a plasmid documented as a genomic region in a previous report. A number of mobile genetic elements (MGEs), including 78 insertion sequences, six genomic islands, and six prophages, were identified in the genome. A putative lysozyme-encoding gene from prophage P6 was shown to express lysin which caused cell lysis. Analysis of the methylome and genome uncovered a pair of reverse-complementary DNA methylation motifs which were widespread in the genome, as well as genes potentially encoding their cognate type I restriction-modification system PpoAI. Further genetic analysis confirmed the function of PpoAI as a RM system in modifying and restricting DNA. The average frequency of the DNA methylation motifs in MGEs was lower than that in the genome, implicating a role of PpoAI in restricting MGEs during genomic evolution of P. polymyxa. Finally, comparative analysis of R, M, and S subunits of PpoAI showed that homologs of the PpoAI system were widely distributed in species belonging to other classes of Firmicute, implicating a role of the ancestor of PpoAI in the genomic evolution of species beyond Paenibacillus.

show abstract