Philip Matsumura

Increased Motility of Escherichia coli by Insertion Sequence Element Integration into the Regulatory Region of the flhD Operon

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.

FlhD/FlhC Is a Regulator of Anaerobic Respiration and the Entner-Doudoroff Pathway through Induction of the Methyl-Accepting Chemotaxis Protein Aer

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated. Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increased gltBD, gcvTHP, and ompT expression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.

Crystal Structures of CheY Mutants Y106W and T87I/Y106W CheY ACTIVATION CORRELATES WITH MOVEMENT OF RESIDUE 106

Position 106 in CheY is highly conserved as an aromatic residue in the response regulator superfamily. In the structure of the wild-type, apo-CheY, Tyr106 is a rotamer whose electron density is observed in both the inside and the outside positions. In the structure of the T87I mutant of CheY, the threonine to isoleucine change at position 87 causes the side chain of Tyr106 to be exclusively restricted to the outside position. In this report we demonstrate that the T87I mutation causes cells to be smooth swimming and non-chemotactic. We also show that another CheY mutant, Y106W, causes cells to be more tumbly than wild-type CheY, and impairs chemotaxis. In the structure of Y106W, the side chain of Trp106 stays exclusively in the inside position. Furthermore, a T87I/Y106W double mutant, which confers the same phenotype as T87I, restricts the side chain of Trp106 to the outside position. The results from these behavioral and structural studies indicate that the rotameric nature of the Tyr106 residue is involved in activation of the CheY molecule. Specifically, CheYs signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain. Our data also suggest that these mutations affect the signal at an event subsequent to phosphorylation.

Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY-phosphate.

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheAL and CheAS. CheAL plays a central role in the Escherichiacoli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheAS remain unknown.

Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli

The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FliA protein, is an alternative ß factor (ß28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of ß28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both ß70 RNA polymerase holoenzyme with FlhD/C (Eß70‐FlhD/C) and ß28 RNA polymerase holoenzyme (Eß28). The flhB operon could only be transcribed by Eß70‐FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by Eß28 could outcompete that by Eß70‐FlhD/C, indicating a positive autoregulation. However, Eß28 could not displace Eß70‐FlhD/C bound to the fliL promoter. The c28‐mediated positive regulation of the class II operons involved a mechanism in which ß28 competed with ß70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the ß70 ‐10 site to the ß28 ‐10 was facilitated by formation of Eß70‐FlhD/C pre‐initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by ß28. We propose that class II operons may be further divided into different subcategories.

An alternative sigma factor controls transcription of flagellar class-III operons in Escherichia coli: gene sequence, overproduction, purification and characterization

Based on the studies of the FliA protein in Bacillus subtilis (Bs) and Salmonella typhimurium (St), the Escherichia coli (Ec) fliA gene has been proposed to encode a flagellar-specific sigma factor, sigma 28. In this study, the complete nucleotide (nt) sequence of Ec fliA was determined. The fliA coding region consists of 717 nt starting with a GTG start codon and ending with a TAA stop codon. The gene product is predicted to be 239 amino acids (26,435 Da). Sequence comparison between Ec FliA and the sigma 28 of St revealed 93.7% identity. Gene fliA was amplified by the polymerase chain reaction, subcloned into expression vector pT7-7, and overexpressed. The overproduced 28-kDa FliA protein, recognized by the St anti-sigma 28 antibody, was purified to homogeneity. The purified protein was able to initiate transcription from the tar promoter in the presence of RNP core enzyme. We conclude that FliA functions as an alternative sigma factor sigma 28 which is specific for flagellar operons in Ec.

Use of a computer to assay motility in bacteria

An assay was developed to study the movement of free-swimming Escherichia coli. Cells were videotaped through a microscope, and the videotape images were then digitized and analyzed with a computer. Angular and linear speeds were measured for wild-type E. coli and for a smooth and a tumbly mutant. The average angular and linear speeds of a population were directly and inversely proportional, respectively, to the time spent tumbling. Changes in angular and linear speeds were followed during the response of wild-type E. coli to attractant or repellent.

Mutations Leading to Altered CheA Binding Cluster on a Face of CheY

CheY is the response regulator of Escherichiacoli chemotaxis and is one of the best studied response regulators of the two-component signaling system. CheY can receive phosphate from the histidine kinase, CheA. Phospho-CheY interacts with the motor-switch complex to induce clockwise flagellar rotation, thus causing the cell to tumble. We used an enzyme-linked immunosorbent assay to study the direct interaction between the kinase, CheA, and the regulator, CheY. The products of random, suppressor, and site-specific cheY mutants were assayed for their ability to bind CheA. Nine mutants showed altered binding. We sequenced and mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding. Interestingly, five of these altered binding mutants were previously defined as being involved in motor-switch binding interactions. This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY. Using CheY (Trp-58) fluorescence quenching, we determined the equilibrium dissociation constants of CheA(124-257) binding for these CheY mutants. The results from the fluorescence quenching are in close agreement with our initial enzyme-linked immunosorbent assay results. Therefore, we propose that the CheA and the motor binding surfaces on CheY partially overlap and that this overlap allows CheY to interact with either the CheA or the flagellar motor, depending on its signaling (phosphorylation) state.

Flagellar Motor-switch Binding Face of CheY and the Biochemical Basis of Suppression by CheY Mutants That Compensate for Motor-switch Defects in Escherichia coli

CheY is a response regulator protein ofEscherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheYwith limited allele specificity. The protein products of suppressorcheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called “pseudotaxis.” Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.

Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG

The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation. The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN. During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system. CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown. We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch. We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein. The sequence data revealed a 91.8% amino acid (aa) identity between E. coli and S. typhimurium FliG. Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector. FliG was successfully overproduced and purified to > 98% purity. Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell. Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.