gE, an RNA polymerase ofactor ofapparent Mr 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarase gene (dagA) was grown in YEME medium containing 34% sucrose and 5 mM MgCl2 to OD6oo = 0.6-0.8, and o-E-containing RNA polymerase holoenzyme was purified from approximately 50 g of mycelium as described (2,4). In vitro transcription reactions on the dagA promoter region carried on a 589-bp Sma I-Ava II DNA fragment (4) were performed as described previously (2, 4) and oE was renatured for holoenzyme reconstitution experiments by using the GroEL-assisted protocol ofBrown et al. (2). Fractions ofRNA polymerase eluted from the final Superose 6 (Pharmacia) gel filtration column with peak levels of dagA P2 transcribing activity were pooled, electrophoresed on a 7.5% polyacrylamide/SDS gel, and electroblotted for 30 min at 0.5 A onto Trans-Blot [Trans-Blot Transfer Medium polyvinylidene difluoride (PVDF) protein sequencing membrane; Bio-Rad] in 10 mM cyclohexylaminopropanesulfonic acid (Caps), pH 11.0/10%o (vol/vol) aqueous methanol. The membrane was subsequently stained and treated as described (2) to visualize the yE polypeptide. The sequence of the first 27 residues of aE was determined by sequential N-terminal Edman degradation using a gas-phase sequenator.Cloning and Nucleotide Sequencing of the sigE Gene. The 256-fold-degenerate 49-mer G332, designed from the sequence of the first 17 N-terminal residues of o-E, was

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methylaccepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.Aerotaxis, the movement of a cell or organism toward or away from oxygen, was first described in bacteria more than a century ago by Engelmann (8), Pfeffer (24), and Beijerinck (3), who observed accumulation of cells near air bubbles or other sources of oxygen. Despite considerable study, particularly in Escherichia coli (22,28,30), the molecular mechanism underlying this behavior has remained elusive. Does the organism detect oxygen directly, or does it instead sense some metabolic consequence of different oxygen environments, such as changes in electron transport activity (19), cellular redox potential (4), or proton motive force (20, 32)? We describe here a gene, dubbed aer for aerotaxis, that encodes a likely flavoprotein signal transducer for aerotaxis in E. coli. Studies of the Aer protein promise to provide a definitive answer to the longstanding puzzle of how cells detect oxygen gradients during aerotaxis.Sequence features of the aer locus. We initially identified the aer gene as an open reading frame (ORF506) discovered in the E. coli genome sequencing project (7). Its conceptual translation product, a 506-amino-acid Aer protein, exhibits several hallmarks of an aerosensing function (Fig. 1). Aer residues 10 to 110 are similar to parts of NifL, FixL, and related bacterial proteins that trigger regulatory responses to changes in environmental oxygen levels (5, 11). Residues 168 to 209 are predominantly hydrophobic and could serve to anchor Aer in the cytoplasmic membrane. Aer residues 259 to 506 are about 50% identical to the cytoplasmic signaling domains of methyl-accepting chemotaxis proteins (MCPs), the principal chemoreceptors of E. coli (14). These features suggested that Aer might generate chemotactic signals in response to oxygen gradients.Construction of an aer mutant. We constructed a large inframe deletion lacking codons 5 to 505 of the aer coding region by PCR amplification of chromosomal sequences flanking the aer locus in strain RP437 (23) by using primer pairs NSB19-NSB20 and NSB25-NSB22 (Fig. 1). The two PCR fragments were ligated at their common XbaI site, joining aer codon 4 to codon 506, and inserted into the pMAK705 vector, whose replication is temperature-sensitive (13), producing plasmid pSB25. RP437 carrying pSB25 was grown at 44°C to select recombinational insertions and then at 30°C for recombin...

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32‐regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5′ and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E‐dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady‐state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

SummaryThe correct annotation of genes encoding the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short open reading frames have been confirmed to correspond to synthesized proteins. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16-50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated genes by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. We summarize the properties of these small proteins; remarkably more than half of the proteins are predicted to be single-transmembrane proteins, nine of which we show co-fractionate with cell membranes.