Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in Escherichia coli

ABSTRACT We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.

[1]  D. Lipman,et al.  A genomic perspective on protein families. , 1997, Science.

[2]  E V Koonin,et al.  How many genes can make a cell: the minimal-gene-set concept. , 2000, Annual review of genomics and human genetics.

[3]  D. Court,et al.  30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15. , 2006, RNA.

[4]  M. Gottesman,et al.  Lytic Mode of Lambda Development , 1983 .

[5]  D. Court,et al.  A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants. , 2003, Biochemistry.

[6]  J Dennis,et al.  Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis , 1999, Molecular microbiology.

[7]  A. Wada,et al.  Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[8]  J. W. Campbell,et al.  Experimental Determination and System Level Analysis of Essential Genes in Escherichia coli MG1655 , 2003, Journal of bacteriology.

[9]  V. Ramakrishnan,et al.  Ribosome Structure and the Mechanism of Translation , 2002, Cell.

[10]  E. Dabbs,et al.  Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties. , 1991, Biochimie.

[11]  D. Court,et al.  Requirement for NusG for transcription antitermination in vivo by the lambda N protein. , 2002, Journal of Bacteriology.

[12]  B. C. Persson,et al.  Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics , 1995, Journal of bacteriology.

[13]  D. Court,et al.  Requirement for NusG for Transcription Antitermination In Vivo by the λ N Protein , 2002 .

[14]  Poul Nissen,et al.  The social life of ribosomal proteins , 2005, The FEBS journal.

[15]  C. Squires,et al.  In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination , 2004, Journal of bacteriology.

[16]  N. Costantino,et al.  Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination , 2003 .

[17]  M. Nomura,et al.  Assembly of bacterial ribosomes. , 1972, Federation proceedings.

[18]  Akira Wada,et al.  Escherichia coli Ribosome-Associated Protein SRA, Whose Copy Number Increases during Stationary Phase , 2001, Journal of bacteriology.

[19]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[20]  B. Bochner,et al.  Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. , 2001, Genome research.

[21]  C. Squires,et al.  An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Joyce Li,et al.  Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription. , 1993, Genes & development.

[23]  N. Costantino,et al.  A set of recombineering plasmids for gram-negative bacteria. , 2006, Gene.

[24]  C W Hill,et al.  Inversions between ribosomal RNA genes of Escherichia coli. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C. Squires,et al.  Antiterminator‐dependent modulation of transcription elongation rates by NusB and NusG , 1999, Molecular microbiology.

[26]  H. Mori,et al.  Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli , 2005, Genes to cells : devoted to molecular & cellular mechanisms.

[27]  G. Culver.  Assembly of the 30S ribosomal subunit. , 2003, Biopolymers.

[28]  D. Friedman,et al.  Reduced Rho-dependent transcription termination permits NusA-independent growth of Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Gottesman,et al.  Requirement for E. coli NusG protein in factor-dependent transcription termination , 1992, Cell.

[30]  M. Deutscher,et al.  Exoribonucleases and their multiple roles in RNA metabolism. , 2001, Progress in nucleic acid research and molecular biology.

[31]  A. Serganov,et al.  Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation , 2004, Molecular microbiology.

[32]  A. Wada.  Growth phase coupled modulation of Escherichia coli ribosomes , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[33]  J. Lupski,et al.  Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in Escherichia coli , 1998, Molecular microbiology.

[34]  D. Friedman,et al.  Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes , 1991, Journal of bacteriology.

[35]  J. Greenblatt,et al.  Recognition of boxA antiterminator RNA by the E. coli antitermination factors NusB and ribosomal protein S10 , 1993, Cell.

[36]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  L. Isaksson,et al.  Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20 , 1993, Molecular microbiology.

[38]  Koreaki Ito,et al.  Insertional disruption of the nusB (ssyB) gene leads to cold-sensitive growth of Escherichia coli and suppression of the secY24 mutation , 1992, Molecular and General Genetics MGG.

[39]  K. Jensen,et al.  NusA Is Required for Ribosomal Antitermination and for Modulation of the Transcription Elongation Rate of both Antiterminated RNA and mRNA* , 1997, The Journal of Biological Chemistry.

[40]  J. Greenblatt,et al.  Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[41]  M. Sørensen,et al.  Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo. , 1998, Journal of molecular biology.

[42]  J. Roberts.  RNA and protein elements of E. coli and lambda transcription antitermination complexes. , 1993, Cell.

[43]  C. Hutchison,et al.  Essential genes of a minimal bacterium. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[44]  C. Squires,et al.  Ribosomal RNA operon anti-termination. Function of leader and spacer region box B-box A sequences and their conservation in diverse micro-organisms. , 1989, Journal of molecular biology.

[45]  A. Spirin,et al.  The ribosome-associated inhibitor A reduces translation errors. , 2004, Biochemical and biophysical research communications.

[46]  Daniel N. Wilson,et al.  Ribosomal Proteins in the Spotlight , 2005, Critical reviews in biochemistry and molecular biology.

[47]  K. Reich.  The search for essential genes. , 2000, Research in microbiology.

[48]  B. Nichols,et al.  Sequence Analysis of Tn10 Insertion Sites in a Collection of Escherichia coli Strains Used for Genetic Mapping and Strain Construction , 1998, Journal of bacteriology.

[49]  H. Noller,et al.  Creating ribosomes with an all-RNA 30S subunit P site. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. Lupski,et al.  Chromosomal duplications in bacteria, fruit flies, and humans. , 1996, American journal of human genetics.

[51]  A. Miczak,et al.  RNA processing in prokaryotic cells , 1993, BioEssays : news and reviews in molecular, cellular and developmental biology.

[52]  R. Green,et al.  Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation. , 2005, Journal of molecular biology.

[53]  D Court,et al.  Isolation and characterization of conditional lethal mutants of Escherichia coli defective in transcription termination factor rho. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[54]  D. Court,et al.  Genetic engineering using homologous recombination. , 2002, Annual review of genetics.

[55]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[56]  C. Squires,et al.  Proteins shared by the transcription and translation machines. , 2000, Annual review of microbiology.

[57]  D. Court,et al.  Transcription antitermination: the λ paradigm updated , 1995 .

[58]  N. Moran,et al.  Microbial Minimalism Genome Reduction in Bacterial Pathogens , 2002, Cell.

[59]  D. Oliver,et al.  ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB , 1994, Journal of bacteriology.

[60]  A. Grossman,et al.  A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. , 1989, Microbiological reviews.

[61]  J. Richardson.  Rho-dependent termination and ATPases in transcript termination. , 2002, Biochimica et biophysica acta.

[62]  T. A. Krulwich,et al.  Identification of a putative Bacillus subtilis rho gene , 1993, Journal of bacteriology.

[63]  Yoshikazu Nakamura,et al.  Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda , 1986, Molecular and General Genetics MGG.

[64]  D. Court,et al.  Genetic analysis of the rnc operon of Escherichia coli , 1989, Journal of bacteriology.

[65]  D. Court,et al.  Transcription antitermination: the lambda paradigm updated. , 1995, Molecular microbiology.

[66]  C. Squires,et al.  Transcriptional Polarity in rRNA Operons of Escherichia coli nusA and nusB Mutant Strains , 2005, Journal of bacteriology.

[67]  J. Mekalanos,et al.  Transposon-based approaches to identify essential bacterial genes. , 2000, Trends in microbiology.

[68]  C. Burns,et al.  Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli. , 1998, Journal of molecular biology.

[69]  A. Ishihama,et al.  Ribosome modulation factor: stationary growth phase-specific inhibitor of ribosome functions from Escherichia coli. , 1995, Biochemical and biophysical research communications.

[70]  N. Fujita,et al.  Regulation of the Escherichia coli rmf gene encoding the ribosome modulation factor: growth phase‐ and growth rate‐dependent control. , 1993, The EMBO journal.

[71]  M. Rosenberg,et al.  rho Is Not Essential for Viability or Virulence inStaphylococcus aureus , 2001, Antimicrobial Agents and Chemotherapy.

[72]  Jeffrey W. Roberts.  RNA and protein elements of E. coli and λ transcription antitermination complexes , 1993, Cell.

[73]  A. Spirin,et al.  A novel stress-response protein that binds at the ribosomal subunit interface and arrests translation. , 2001, Cold Spring Harbor symposia on quantitative biology.

[74]  D. Court,et al.  High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[75]  G. Church,et al.  Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization , 1997, Journal of bacteriology.