title: Mutations in the primary sigma factor σA and termination factor rho that reduce susceptibility to cell wall antibiotics.
abstract: Combinations of glycopeptides and β-lactams exert synergistic antibacterial activity, but the evolutionary mechanisms driving resistance to both antibiotics remain largely unexplored. By repeated subculturing with increasing vancomycin (VAN) and cefuroxime (CEF) concentrations, we isolated an evolved strain of the model bacterium Bacillus subtilis with reduced susceptibility to both antibiotics. Whole-genome sequencing revealed point mutations in genes encoding the major σ factor of RNA polymerase (sigA), a cell shape-determining protein (mreB), and the ρ termination factor (rho). Genetic-reconstruction experiments demonstrated that the G-to-C substitution at position 336 encoded by sigA (sigA(G336C)), in the domain that recognizes the -35 promoter region, is sufficient to reduce susceptibility to VAN and works cooperatively with the rho(G56C) substitution to increase CEF resistance. Transcriptome analyses revealed that the sigA(G336C) substitution has wide-ranging effects, including elevated expression of the general stress σ factor (σ(B)) regulon, which is required for CEF resistance, and decreased expression of the glpTQ genes, which leads to fosfomycin (FOS) resistance. Our findings suggest that mutations in the core transcriptional machinery may facilitate the evolution of resistance to multiple cell wall antibiotics.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York, USA Department of Biomedical Laboratory Science, Dongseo University, Busan, South Korea.
authors.forename: Yong Heon
authors.initials: YH
authors.lastname: Lee
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York, USA jdh9@cornell.edu.
authors.forename: John D
authors.initials: JD
authors.lastname: Helmann
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D005075:Biological Evolution; D002444:Cefuroxime / Q000494:pharmacology; D002473:Cell Wall / Q000187:drug effects*; D024881:Drug Resistance, Bacterial / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016680:Genome, Bacterial; D009154:Mutation; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D010454:Peptide Termination Factors; D012808:Sigma Factor; D014640:Vancomycin; D002444:Cefuroxime
references.citation: Cell. 2003 Jul 11;114(1):135-46
references.pmid: 12859904
references.citation: Microb Inform Exp. 2013 Apr 10;3(1):2
references.pmid: 23575213
references.citation: J Biol Chem. 2001 Jul 27;276(30):28380-7
references.pmid: 11369775
references.citation: J Mol Biol. 1995 Oct 6;252(5):536-49
references.pmid: 7563072
references.citation: EMBO Mol Med. 2013 Apr;5(4):486-7
references.pmid: 23554168
references.citation: Lancet Infect Dis. 2005 Feb;5(2):115-9
references.pmid: 15680781
references.citation: Genome Biol. 2003;4(1):203
references.pmid: 12540296
references.citation: Genes Dev. 1993 Jan;7(1):149-60
references.pmid: 8422984
references.citation: J Bacteriol. 2000 Aug;182(16):4478-90
references.pmid: 10913081
references.citation: Antimicrob Agents Chemother. 2001 Apr;45(4):1099-103
references.pmid: 11257021
references.citation: Annu Rev Microbiol. 2003;57:441-66
references.pmid: 14527287
references.citation: Mol Microbiol. 1999 Jan;31(2):651-63
references.pmid: 10027981
references.citation: Antimicrob Agents Chemother. 2013 Jan;57(1):56-65
references.pmid: 23070162
references.citation: Mol Cell. 2001 Jul;8(1):21-31
references.pmid: 11511357
references.citation: J Bacteriol. 2001 Oct;183(20):5918-26
references.pmid: 11566991
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D225-9
references.pmid: 21109532
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6482-6
references.pmid: 20308541
references.citation: Antimicrob Agents Chemother. 1999 Jul;43(7):1747-53
references.pmid: 10390234
references.citation: Antimicrob Agents Chemother. 2011 Sep;55(9):4326-37
references.pmid: 21709092
references.citation: Mol Microbiol. 2008 Feb;67(4):830-48
references.pmid: 18179421
references.citation: Curr Opin Struct Biol. 2003 Feb;13(1):31-9
references.pmid: 12581657
references.citation: J Biol Chem. 1996 Oct 11;271(41):25369-74
references.pmid: 8810302
references.citation: Mol Microbiol. 2012 Feb;83(3):623-39
references.pmid: 22211522
references.citation: Mol Microbiol. 2002 Sep;45(5):1267-76
references.pmid: 12207695
references.citation: Annu Rev Biochem. 1983;52:825-69
references.pmid: 6351730
references.citation: J Mol Biol. 2011 Oct 7;412(5):793-813
references.pmid: 21439297
references.citation: J Bacteriol. 1997 Aug;179(16):5238-40
references.pmid: 9260971
references.citation: Infect Genet Evol. 2014 Jan;21:575-82
references.pmid: 23567819
references.citation: J Bacteriol. 2001 Oct;183(19):5617-31
references.pmid: 11544224
references.citation: Annu Rev Microbiol. 2007;61:215-36
references.pmid: 18035607
references.citation: Microbiology (Reading). 2012 Mar;158(Pt 3):696-707
references.pmid: 22174379
references.citation: Nat Rev Microbiol. 2009 Jun;7(6):460-6
references.pmid: 19444248
references.citation: PLoS Genet. 2008 Aug 01;4(8):e1000139
references.pmid: 18670626
references.citation: J Bacteriol. 2001 Nov;183(22):6573-8
references.pmid: 11673427
references.citation: FEMS Microbiol Rev. 2008 Mar;32(2):234-58
references.pmid: 18266856
references.citation: Pharmacol Rev. 1989 Jun;41(2):93-141
references.pmid: 2692037
references.citation: Mol Microbiol. 1995 Dec;18(5):943-52
references.pmid: 8825098
references.citation: Eur J Clin Microbiol Infect Dis. 1989 Nov;8(11):943-50
references.pmid: 2532132
references.citation: Mol Microbiol. 2003 Dec;50(5):1591-604
references.pmid: 14651641
references.citation: J Bacteriol. 1993 Feb;175(3):647-54
references.pmid: 8423140
references.citation: Res Microbiol. 2002 Nov;153(9):557-62
references.pmid: 12455702
references.citation: Antimicrob Agents Chemother. 2006 Nov;50(11):3622-30
references.pmid: 16954318
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13977-81
references.pmid: 18779569
references.citation: J Bacteriol. 1994 Aug;176(16):5033-43
references.pmid: 8051015
references.citation: J Bacteriol. 1978 Feb;133(2):667-70
references.pmid: 415047
references.citation: Curr Opin Microbiol. 1999 Oct;2(5):489-93
references.pmid: 10508723
references.citation: Appl Environ Microbiol. 2008 Jun;74(11):3419-25
references.pmid: 18408063
references.citation: Proc Natl Acad Sci U S A. 1976 Jun;73(6):1959-63
references.pmid: 132662
references.citation: Science. 2008 Jul 18;321(5887):356-61
references.pmid: 18635788
references.citation: Genome Biol. 2007;8(1):201
references.pmid: 17274841
references.citation: Antimicrob Agents Chemother. 2004 Aug;48(8):2888-96
references.pmid: 15273097
references.citation: J Bacteriol. 2001 Dec;183(24):7318-28
references.pmid: 11717291
references.citation: Biochemistry. 1994 Jul 12;33(27):8292-9
references.pmid: 7518246
references.citation: Biochim Biophys Acta. 2011 Dec;1810(12):1323-9
references.pmid: 21791237
references.citation: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9451-6
references.pmid: 17517606
references.citation: J Mol Biol. 1995 Sep 15;252(2):189-202
references.pmid: 7545758
references.citation: PLoS Genet. 2012 May;8(5):e1002744
references.pmid: 22693458
references.citation: Biochim Biophys Acta. 2002 Sep 13;1577(2):251-260
references.pmid: 12213656
references.citation: Proc Natl Acad Sci U S A. 1985 Apr;82(7):1911-5
references.pmid: 2580303
references.citation: Mol Microbiol. 2001 Aug;41(4):757-74
references.pmid: 11532142
references.citation: Nat Genet. 2006 Apr;38(4):489-94
references.pmid: 16550172
references.citation: Science. 2012 Mar 2;335(6072):1103-6
references.pmid: 22383849
references.citation: J Bacteriol. 2004 Jun;186(12):4034-7
references.pmid: 15175319
references.citation: Semin Respir Crit Care Med. 2006 Feb;27(1):68-81
references.pmid: 16508883
references.citation: J Antimicrob Chemother. 2013 Mar;68(3):496-505
references.pmid: 23134656
references.citation: Cell. 2006 Nov 3;127(3):553-64
references.pmid: 17081977
references.citation: J Mol Biol. 1994 May 27;239(1):1-14
references.pmid: 7515111
references.citation: J Clin Microbiol. 2011 Jul;49(7):2680-4
references.pmid: 21525224
references.citation: J Bacteriol. 2001 Apr;183(7):2380-3
references.pmid: 11244082
references.citation: Antimicrob Agents Chemother. 2010 Dec;54(12):5222-33
references.pmid: 20837752
references.citation: Microbiology (Reading). 1994 Apr;140 ( Pt 4):723-30
references.pmid: 8012593
references.citation: Proc Natl Acad Sci U S A. 2010 Nov 23;107(47):20500-5
references.pmid: 21057108
references.citation: Antimicrob Agents Chemother. 2009 Apr;53(4):1598-609
references.pmid: 19164152
references.citation: Antimicrob Agents Chemother. 2006 Jun;50(6):2137-45
references.pmid: 16723576
references.citation: J Bacteriol. 2013 Jan;195(1):156-66
references.pmid: 23104812