Inactivation of Chromosomal Genes in Serratia marcescens

Kamaletdinova, Leisan Kh.; Nizamutdinova, E. Kh.; Shirshikova, Tatiana V.; Skipina, Irina M.; Bogomolnaya, Lydia M.

doi:10.1007/s12668-016-0249-2

Cited by 3 publications

(6 citation statements)

References 11 publications

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“…All S. marcescens strains used in this study are listed in Table 3 . The Δ macAB mutant strain was generated by lambda red homologous recombination ( 74 , 75 ) in nuclease-deficient, restrictionless S. marcescens strain TT392 ( 76 ), and the mutation was then moved into S. marcescens SM6 by bacteriophage ΦOT8 transduction ( 77 , 78 ). Strains were routinely grown in LB broth (10 g/liter Bacto-Tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) or Mueller-Hinton broth (BD Difco).…”

Section: Methodsmentioning

confidence: 99%

“…The complementation plasmid carrying intact macAB genes with C-terminal 6×His tag was generated as follows: S. marcescens strain with chromosomal macB-6×His fusion (LMB184) was generated using the previously established protocol ( 74 , 79 ) with primers macB-6×His-FWD 5′-TGCCGCGCGGCTGAATCCGATCGATGCGCTGGCGCGCGAGCACCACCATCATCACCATTAGT-3′ and macB-tagging-REV 5′-TGCCAGCCGCCGTGTGACTGGCATTTTTTATGCCTTTTACTATGAATATCCTCCTTAG-3′ and the template plasmid pSUB7 ( 79 ). A DNA fragment containing the full-length open reading frame with 247 bp upstream and 183 bp downstream of macAB was amplified using genomic DNA from LMB184 by PCR with macA-FWD-SacI 5′-AGCGAGCTCTGACATGATGAAATCCTT-3′ as a forward primer and macB-REV-KpnI 5′-ATGGTACCAAGGCGGGCCAGCAGGTC-3′ as a reverse primer.…”

Section: Methodsmentioning

confidence: 99%

“…The S. marcescens strain with chromosomal macA-FLAG fusion (LB485) was generated with primers macA-FLAG-FWD 5′-CGATGAGGTGATCGTCAGCCGCGGCGGCGTGGAGGCCGGCGACTACAAAGATGACGACGATAAATAG-3′ and macA-tagging-REV 5′-TAGCTGCGGCGAATGCCGTTCAGCTGCAACAGCGCCGCCATATGAATATCCTCCTTAG-3′ and the template plasmid pSU313 using the previously established protocol ( 74 , 79 ). The resulting strain was used for PCR amplification of the macA coding sequence with 247 bp upstream and 119 bp downstream using primers macA-FWD-SacI as a forward primer and macA-REV-KpnI 5′-ATGGTACCCATGATCGCCACCATTTC-3′ as a reverse primer.…”

Section: Methodsmentioning

confidence: 99%

“…The S. marcescens strain with chromosomal macB -FLAG fusion (LMB163) was generated using a previously established protocol ( 74 , 79 ). Briefly, primers macB-FLAG-FWD 5′-TGCCGCGCGGCTGAATCCGATCGATGCGCTGGCGCGCGAGGACTACAAAGATGACGACGATAAATAG-3′ and macB-tagging-REV were used to amplify antibiotic-resistant cassette from the template plasmid pSU312 ( 79 ).…”

Section: Methodsmentioning

confidence: 99%

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The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress

Shirshikova

Sierra-Bakhshi

Kamaletdinova

et al. 2021

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Serratia marcescens is an emerging pathogen with increasing clinical importance due to its intrinsic resistance to several classes of antibiotics. The chromosomally encoded drug efflux pumps contribute to antibiotic resistance and represent a major challenge for the treatment of bacterial infections. The ABC-type efflux pump MacAB was previously linked to macrolide resistance in Escherichia coli and Salmonella enterica serovar Typhimurium. The role of the MacAB homolog in antibiotic resistance of S. marcescens is currently unknown. We found that an S. marcescens mutant lacking the MacAB pump did not show increased sensitivity to the macrolide antibiotic erythromycin but was significantly more sensitive to aminoglycoside antibiotics and polymyxins. We also showed that, in addition to its role in drug efflux, the MacAB efflux pump is required for swimming motility and biofilm formation. We propose that the motility defect of the ΔmacAB mutant is due, at least in part, to the loss of functional flagella on the bacterial surface. Furthermore, we found that the promoter of the MacAB efflux pump was active during the initial hours of growth in laboratory medium and that its activity was further elevated in the presence of hydrogen peroxide. Finally, we demonstrate a complete loss of ΔmacAB mutant viability in the presence of peroxide, which is fully restored by complementation. Thus, the S. marcescens MacAB efflux pump is essential for survival during oxidative stress and is involved in protection from polymyxins and aminoglycoside antibiotics. IMPORTANCE The opportunistic pathogen Serratia marcescens can cause urinary tract infections, respiratory infections, meningitis, and sepsis in immunocompromised individuals. These infections are challenging to treat due to the intrinsic resistance of S. marcescens to an extensive array of antibiotics. Efflux pumps play a crucial role in protection of bacteria from antimicrobials. The MacAB efflux pump, previously linked to efflux of macrolides in Escherichia coli and protection from oxidative stress in Salmonella enterica serovar Typhimurium, is not characterized in S. marcescens. We show the role of the MacAB efflux pump in S. marcescens protection from aminoglycoside antibiotics and polymyxins, modulation of bacterial motility, and biofilm formation, and we illustrate the essential role for this pump in bacterial survival during oxidative stress. Our findings make the MacAB efflux pump an attractive target for inhibition to gain control over S. marcescens infections.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress

Shirshikova

Sierra-Bakhshi

Kamaletdinova

et al. 2021

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“…As a matter of fact, one of the most prominent advantages of Red recombineering is that homologous arms as short as 35 bp can work efficiently in genome editing, which opens a vast array of new possibilities for generating recombinant DNA and makes the modification procedure extremely simple [19,20]. Indeed, there is one report about short homologous arms successfully used for recombination in S. marcescens, but the host genome needs to be modified in advance [27].…”

Section: Introductionmentioning

confidence: 99%

Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

Chen

Cao

2021

Appl Biochem Biotechnol

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Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCRamplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible P BAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, Gm R -kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.

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Serratia marcescens DUF1471-Containing Protein SrfN Is Needed for Adaptation to Acid and Oxidative Stresses

Elistratova

Matrosova

Khilyas

et al. 2022

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Inactivation of Chromosomal Genes in Serratia marcescens

Cited by 3 publications

References 11 publications

The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress

The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress

Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

Serratia marcescens DUF1471-Containing Protein SrfN Is Needed for Adaptation to Acid and Oxidative Stresses

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