Carol A. Gross

From the regulation of peptidoglycan synthesis to bacterial growth and morphology

How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.

Mapping and sequencing of mutations in the Escherichia colirpoB gene that lead to rifampicin resistance

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.

Is hsp70 the cellular thermometer

Cells respond to an increase in temperature by inducing the synthesis of the heat shock proteins, which are a small set of evolutionarily conserved proteins. We review the evidence leading us to suggest that the free pool of one of these proteins, hsp70, serves as a cellular thermometer that regulates the expression of all heat shock proteins.

Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources

Quantitative views of cellular functions require precise measures of rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here, we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli data set transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. Members of multiprotein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles for optimizing design. These include how the level of different types of transcription factors is optimized for rapid response and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. Our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from quantitative analyses of protein synthesis.

OMP Peptide Signals Initiate the Envelope-Stress Response by Activating DegS Protease via Relief of Inhibition Mediated by Its PDZ Domain

Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E). Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription. These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs. DegS activation involves relief of inhibitory interactions between its PDZ and protease domains. Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

The htpR gene product of E. coli is a sigma factor for heat-shock promoters

The htpR gene of E. coli encodes a positive regulator of the heat-shock response. We have fused the htpR gene to the inducible PL promoter of phage lambda. Overproduction of HtpR following a temperature upshift resulted in the overexpression of heat-shock proteins. We describe the purification and initial in vitro characterization of the factor controlling expression of heat-shock genes. The factor was the 32 kd htpR gene product. In vitro, a mixture of HtpR and core RNA polymerase initiated transcription at heat-shock promoters. The sigma factor encoded by rpoD was not required for this reaction. Therefore, HtpR is a sigma factor that promotes transcription initiation at heat-shock promoters. We propose that htpR be renamed rpoH and that the gene product be called sigma-32.

Phenotypic landscape of a bacterial cell.

The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.

The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, σE. σE activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that σE activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25‐fold induction of σE activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits σE‐directed transcription in vitroindicating that RseA acts as an anti‐sigma factor. Deletion of rseB leads to a slight induction of σE, indicating that RseB is also a negative regulator of σE. RseB is a periplasmic protein and was found to co‐purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on σE through RseA. Deletion of rseC, in contrast, has no effect on σE activity under steady‐state conditions. Under induction conditions, strains lacking RseB and/or C show wild‐type induction of σE activity, indicating either the presence of multiple pathways regulating σE activity, or the ability of RseA alone to both sense and transmit information to σE.

Altered promoter recognition by mutant forms of the σ70 subunit of Escherichia coli RNA polymerase

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.

Polypeptides containing highly conserved regions of transcription initiation factor σ70 exhibit specificity of binding to promoter DNA

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.