微生物功能基因组学 翻译

The polar clusters. R. sphaeroides cells typically have a chemotaxis cluster at each pole (FIG. 3). These clusters comprise the transmembrane chemoreceptors and the cheop2-encoded proteins CheA2, CheW2 , CheW3 and CheR2. Cluster formation depends on CheA2, CheW2, CheW3 and the chemoreceptors. The cluster senses the periplasmic concentration of chemoeffectors and controls the rate at which CheA2 autophosphorylates. CheA2-P is capable of phosphorylating all of the R. sphaeroides chemotaxis response regulators, meaning that the polar cluster can influence the phosphorylation levels of all of the chemotaxis response regulators in the cell.

The cytoplasmic cluster. The cytoplasmic cluster comprises the cytoplasmic chemoreceptors and the cheop3-encoded proteins CheA3, CheA4, CheW4and CheR3(ReF.73). Cluster formation requires the cytoplasmic chemoreceptors along with CheW4 and either CheA3 or CheA4 (ReF.83). As with the polar chemo-taxis clusters, there are >1,000 chemoreceptors and associated chemosensory proteins in the cytoplasmic cluster. The arrangement of the proteins is unknown, and it is unclear how they form a quaternary complex in the absence of a membrane scaffold. Newly divided wild-type cells typically have a single cytoplasmic cluster located approximately at mid-cell. Unlike the polar chemotaxis clusters, which are automatically segregated at cell division (as each daughter cell inherits one pole from the parent), the cytoplasmic cluster requires a spe-cialized mechanism of protein segregation to ensure that each daughter cell inherits a cluster (FIG. 3) . Before cell division, the single cluster located at mid-cell seems to become two clusters that migrate to about the one-quarter and three-quarter cell positions, one at each position, ensuring that each daughter cell inherits a single cluster. This intriguing mechanism uses PpfA, a protein encoded in cheop3 that is a homologue of the ParA family type I DNA-partitioning ATPases (which are involved in plas-mid and chromosome partitioning). PpfA controls the number and positioning of cytoplasmic clusters. ΔppfA mutant cells have aberrant positioning of the cytoplas-mic cluster and rarely contain more than a single cluster; the two daughter cells inherit either a single cluster or no cluster at all. Although the daughter cell lacking a cluster eventually synthesizes a new one, the delay could explain the reduced chemotaxis phenotype of the ΔppfA mutant.

The cytoplasmic cluster, which is believed to sense the metabolic state of the cell, controls the activity of the two unusual CheA homologues, CheA3and CheA4. CheA4is an N-terminally truncated CheA homologue that lacks the P1 and P2 domains but retains the P3–P5 domains, while CheA 3 has a P1 and a P5 domain separated by a novel 794 amino acid sequence that has phosphatase activity. On stimulation, CheA4phosphorylates the P1 domain of CheA3on His51. Unlike CheA2-P, which can phosphorylate all eight of the chemotaxis responseregulators, CheA3-P acts as a phosphodonor for only CheY1, CheY6 and CheB2 (ReF.75). CheY6and CheB2 are encoded by cheop3 and are essential for normal chemo-taxis; however, CheY1 is encoded by cheop1 and is not expressed in wild-type cells under laboratory conditions. The cytoplasmic cluster is therefore capable of directly influencing the phosphorylation state of only two of the five chemotaxis response regulators that are nor-mally expressed in wild-type cells, CheY6 and CheB2. In addition to its ability to act as a phosphodonor to these response regulators, CheA3 shows specific phosphatase activity towards CheY6-P; this activity depends on the novel 794 amino acid sequence in CheA3(ReF.88). CheA3and CheA4have different receptor-binding (P5) domains;

therefore, independent regulation of the kinase activity of CheA 4 and the phosphatase activity of CheA3 could be crucial for this pathway.The cytoplasmic cluster, which is believed to sense the metabolic state of the cell, controls the activity of the two unusual CheA homologues, CheA3 and CheA4. CheA4is an N-terminally truncated CheA homologue that lacks the P1 and P2 domains but retains the P3–P5 domains, while CheA3 has a P1 and a P5 domain separated by a novel 794 amino acid sequence in CheA3(ReF.88).CheA3 and CheA4have different receptor-binding (P5) domains; therefore, independent regulation of the kinase activity of CheA4 and the phosphatase activity of CheA 3 could be crucial for this pathway.

Role of CheY. Wild-type R. sphaeroides expresses three of its six CheY proteins under laboratory conditions: CheY3, CheY4 and CheY6. CheY6 is essential for chemotaxis, but there is redundancy between CheY3 and CheY4, with only one being required for chemotaxis. In the absence of chemotaxis proteins, the flagellar motor rotates con-tinuously. CheY6-P is capable of stopping the flagellar motor alone, but without CheY3 or CheY4, it is unable to support chemotaxis. All three of these CheY homo-logues can bind the FliM component of the Fla1 flagellar motor in vitro, but the effects of CheY3-P and CheY4-P binding on flagellar rotation are unknown.