Small Stress Response Proteins in Escherichia coli: Proteins Missed by Classical Proteomic Studies

ABSTRACT Proteins of 50 or fewer amino acids are poorly characterized in all organisms. The corresponding genes are challenging to reliably annotate, and it is difficult to purify and characterize the small protein products. Due to these technical limitations, little is known about the abundance of small proteins, not to mention their biological functions. To begin to characterize these small proteins in Escherichia coli, we assayed their accumulation under a variety of growth conditions and after exposure to stress. We found that many small proteins accumulate under specific growth conditions or are stress induced. For some genes, the observed changes in protein levels were consistent with known transcriptional regulation, such as ArcA activation of the operons encoding yccB and ybgT. However, we also identified novel regulation, such as Zur repression of ykgMO, cyclic AMP response protein (CRP) repression of azuC, and CRP activation of ykgR. The levels of 11 small proteins increase after heat shock, and induction of at least 1 of these, YobF, occurs at a posttranscriptional level. These results show that small proteins are an overlooked subset of stress response proteins in E. coli and provide information that will be valuable for determining the functions of these proteins.

[1]  J. Foster,et al.  GadE (YhiE) activates glutamate decarboxylase‐dependent acid resistance in Escherichia coli K‐12 , 2003, Molecular microbiology.

[2]  G. Storz,et al.  Repression of small toxic protein synthesis by the Sib and OhsC small RNAs , 2008, Molecular microbiology.

[3]  Andrey A Mironov,et al.  Comparative genomics of bacterial zinc regulons: Enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[5]  T. Atlung,et al.  Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli , 1996, Journal of bacteriology.

[6]  Sang Yup Lee,et al.  The Escherichia coli Proteome: Past, Present, and Future Prospects , 2006, Microbiology and Molecular Biology Reviews.

[7]  S. Gottesman,et al.  A PhoQ/P‐regulated small RNA regulates sensitivity of Escherichia coli to antimicrobial peptides , 2009, Molecular microbiology.

[8]  M. Inouye,et al.  Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli , 2001, Molecular microbiology.

[9]  T. D. Schneider,et al.  Small membrane proteins found by comparative genomics and ribosome binding site models , 2008, Molecular microbiology.

[10]  Jolyon Holdstock,et al.  Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  G. Storz,et al.  Small RNAs and Small Proteins Involved in Resistance to Cell Envelope Stress and Acid Shock in Escherichia coli: Analysis of a Bar-Coded Mutant Collection , 2009, Journal of bacteriology.

[12]  Torsten Waldminghaus,et al.  FourU: a novel type of RNA thermometer in Salmonella , 2007, Molecular microbiology.

[13]  Torsten Waldminghaus,et al.  RNA thermometers. , 2006, FEMS microbiology reviews.

[14]  E. Laskowska,et al.  The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State* , 2005, Journal of Biological Chemistry.

[15]  Juan Pablo Couso,et al.  Peptides Encoded by Short ORFs Control Development and Define a New Eukaryotic Gene Family , 2007, PLoS biology.

[16]  C. K. Vanderpool,et al.  A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide , 2007, Proceedings of the National Academy of Sciences.

[17]  Milton H. Saier,et al.  Transcriptome Analysis of Crp-Dependent Catabolite Control of Gene Expression in Escherichia coli , 2004, Journal of bacteriology.

[18]  I. Kurtser,et al.  Replication Initiation Proteins Regulate a Developmental Checkpoint in Bacillus subtilis , 2001, Cell.

[19]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[20]  Julio Collado-Vides,et al.  Sigma70 promoters in Escherichia coli: specific transcription in dense regions of overlapping promoter-like signals. , 2003, Journal of molecular biology.

[21]  A. Bairoch,et al.  Low molecular weight proteins: A challenge for post‐genomic research , 1998, Electrophoresis.

[22]  A. Blanc-Potard,et al.  Peptide‐assisted degradation of the Salmonella MgtC virulence factor , 2008, The EMBO journal.

[23]  Julio Collado-Vides,et al.  RegulonDB (version 5.0): Escherichia coli K-12 transcriptional regulatory network, operon organization, and growth conditions , 2005, Nucleic Acids Res..

[24]  Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the alpha subunit of RNA polymerase at class I promoters. , 1999, The Biochemical journal.

[25]  Scott E. Gabriel,et al.  Contributions of Zur-Controlled Ribosomal Proteins to Growth under Zinc Starvation Conditions , 2009, Journal of bacteriology.

[26]  R. Gennis,et al.  Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product , 1990, Journal of bacteriology.

[27]  G. W. Hatfield,et al.  Leucine-responsive Regulatory Protein-DNA Interactions in the Leader Region of the ilvGMEDA Operon of Escherichia coli* , 1996, The Journal of Biological Chemistry.

[28]  M. Inouye,et al.  CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli , 2001, Molecular microbiology.

[29]  A. Wawrzynów,et al.  IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock. , 1996, Biochimie.

[30]  A. Kennedy,et al.  Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA , 2007, Nature Medicine.

[31]  Mark W Maciejewski,et al.  Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis. , 2004, Molecular cell.

[32]  H. E. Umbarger,et al.  Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon , 1980, Journal of bacteriology.

[33]  T. A. Krulwich,et al.  Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4 , 1996, Journal of bacteriology.

[34]  G. Storz,et al.  The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF‐I) protein , 1998, The EMBO journal.

[35]  Mark Gomelsky,et al.  Cyclic Diguanylate Is a Ubiquitous Signaling Molecule in Bacteria: Insights into Biochemistry of the GGDEF Protein Domain , 2005, Journal of bacteriology.

[36]  R. Griffey,et al.  A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome. , 2002, Bio Systems.

[37]  P Berndt,et al.  Enrichment of low abundance proteins of Escherichia coli by hydroxyapatite chromatography , 1999, Electrophoresis.

[38]  D. Swigon,et al.  Catabolite activator protein: DNA binding and transcription activation. , 2004, Current opinion in structural biology.

[39]  Sarah E. Ades,et al.  Regulation by destruction: design of the sigmaE envelope stress response. , 2008, Current opinion in microbiology.

[40]  Dominique Douguet,et al.  HELIQUEST: a web server to screen sequences with specific alpha-helical properties , 2008, Bioinform..

[41]  D. Court,et al.  Host responses influence on the induction of lambda prophage , 2008, Molecular microbiology.

[42]  Takashi Yura,et al.  Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response , 2008, Microbiology and Molecular Biology Reviews.

[43]  W. Wackernagel,et al.  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. , 1995, Gene.

[44]  J. Foster,et al.  Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes. , 2001, Microbiology.

[45]  Dominique Douguet,et al.  HELIQUEST : a web server to screen sequences with specific α-helical properties , 2008 .

[46]  Torsten Waldminghaus,et al.  RNA thermometers are common in α- and γ-proteobacteria , 2005 .

[47]  S. Gottesman,et al.  The Crp-Activated Small Noncoding Regulatory RNA CyaR (RyeE) Links Nutritional Status to Group Behavior , 2008, Journal of bacteriology.

[48]  M. Maurizi,et al.  Turnover of Endogenous SsrA-tagged Proteins Mediated by ATP-dependent Proteases in Escherichia coli* , 2008, Journal of Biological Chemistry.

[49]  J. H. Collins,et al.  Sarcolipin, the "proteolipid" of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31-residue peptide. , 1992, Archives of biochemistry and biophysics.

[50]  G. Storz,et al.  Target prediction for small, noncoding RNAs in bacteria , 2006, Nucleic acids research.

[51]  S. Gottesman,et al.  A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two‐component system , 2009, Molecular microbiology.

[52]  George M. Church,et al.  Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K‐12 , 1997, Electrophoresis.

[53]  M. Dion,et al.  A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA) , 1991, Molecular and General Genetics MGG.

[54]  Alison I. Graham,et al.  Severe Zinc Depletion of Escherichia coli ROLESFORHIGHAFFINITYZINCBINDINGBYZinT,ZINCTRANSPORTAND ZINC-INDEPENDENTPROTEINS * , 2009 .