Mandy J. Ward

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram‐negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the ‘frizzy’ signal‐transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY‐like response‐regulator domains: one is present on FrzE, which also contains a CheA‐like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self‐generated autoattractants. The Frz signal‐transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting‐body development and vegetative swarming.

Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development

Myxococcus xanthus is a Gram‐negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores. Both of these behaviours require multiple Ser/Thr protein kinases. In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M. xanthus. DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE. Recombinant His‐tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid‐resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase. Pph1‐expression was observed under both vegetative and developmental conditions, but peaked during early aggregation. A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation. Under starvation‐induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores. Using the yeast two‐hybrid system, we have observed a strong interaction between Pph1 and the M. xanthus protein kinase Pkn5, a negative effector of development. These results suggest a functional link between a Pkn2‐type protein kinase and a PP2C phosphatase.

Social motility in Myxococcus xanthus requires FrzS, a protein with an extensive coiled‐coil domain

Gliding motility in the developmental bacterium Myxococcus xanthus involves two genetically distinct motility systems, designated adventurous (A) and social (S). Directed motility responses, which facilitate both vegetative swarming and developmental aggregation, additionally require the ‘frizzy’ (Frz) signal transduction pathway. In this study, we have analysed a new gene (frzS), which is positioned upstream of the frzA–F operon. Insertion mutations in frzS caused both vegetative spreading and developmental defects, including ‘frizzy’ aggregates in the FB strain background. The ‘frizzy’ phenotype was previously considered to result only from defective directed motility responses. However, deletion of the frzS gene in an A−S+ motility background demonstrated that FrzS is a new component of the S‐motility system, as the A−frzS double mutant was non‐spreading (A−S−). Compared with known S‐motility mutants, the frzS mutants appear similar to pilT mutants, in that both produce type IV pili, extracellular fibrils and lipopolysaccharide (LPS) O‐antigen, and both agglutinate rapidly in a cohesion assay. The FrzS protein has an unusual domain composition for a bacterial protein. The N‐terminal domain shows similarity to the receiver domains of the two‐component response regulator proteins. The C‐terminal domain is composed of up to 38 heptad repeats (a b c d e f g)38, in which residues at positions a and d are predominantly hydrophobic, whereas residues at positions e and g are predominantly charged. This periodic disposition of specific residues suggests that the domain forms a long coiled‐coil structure, similar to those found in the α‐fibrous proteins, such as myosin. Overexpression of this domain in Escherichia coli resulted in the formation of an unusual striated protein lattice that filled the cells. We speculate on the role that this novel protein could play in gliding motility.

Motility in Myxococcus xanthus and its role in developmental aggregation

The Frz signal transduction system of Myxococcus xanthus was originally thought to be a simple variation of the well-characterized Che system of the enteric bacteria. Recently, however, many additional Frz proteins, along with alternative signal transduction systems, have been discovered. Together these signal transduction pathways coordinate cell-cell behavior, permitting the complex interactions required for developmental aggregation and fruiting body formation.

Chemotactic Responses to Metals and Anaerobic Electron Acceptors in Shewanella oneidensis MR-1

Although a previous study indicated that the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 lacks chemotactic responses to metals that can be used as anaerobic electron acceptors, new results show that this bacterium responds to both Mn(III) and Fe(III). Cells were also shown to respond to another unusual electron acceptor, the humic acid analog anthraquinone-2,6-disulfonate. These results indicate that S. oneidensis is capable of moving towards a number of unusual anaerobic electron acceptors, including some that would normally be insoluble in the environment. Additionally, S. oneidensis was shown to migrate in gradients of several divalent cations under anaerobic conditions. Although responses to the reduced forms of redox-active metals, such as Mn(II) and Fe(II), might indicate that S. oneidensis uses gradients of these metals to locate the insoluble electron acceptors Mn(III/IV) and Fe(III) for dissimilatory purposes, responses to non-redox-active metals, such as Zn(II), suggest that movement towards divalent cations might serve other, potentially assimilatory, purposes.

IDENTIFICATION AND CHARACTERIZATION OF FRZZ, A NOVEL RESPONSE REGULATOR NECESSARY FOR SWARMING AND FRUITING-BODY FORMATION IN MYXOCOCCUS XANTHUS

The frz genes of Myxococcus xanthus constitute a signal‐transduction pathway that processes chemotactic information in a manner analogous to that found in enteric bacteria. Ultimately, these genes regulate the frequency of individual cell reversal. We report here the identification of a novel component of this signal‐transduction pathway, designated frzZ, which was discovered as an open reading frame located 5′ to the frz operon but transcribed in the opposite orientation. The translational start site of frzZ  is 170 base pairs from that of frzAfrzZ  utilizes a promoter similar to the σ70 promoters of Escherichia coli, and encodes a 290‐amino‐acid soluble protein, FrzZ (Mr 30 500). FrzZ contains two domains, both of which show strong homology to CheY and other members of the response‐regulator family. Linking these domains is a 39‐amino‐acid region that is very rich in alanine and proline (38% Ala and 33% Pro). A frzZ null mutant showed abnormally low reversal rates when compared to the wild‐type control and was unable to form fruiting bodies on starvation medium, but it did form ‘frizzy’ aggregates. In addition, the frzZ mutant was defective in swarming, particularly on soft agar (0.3% w/v). However, unlike most frz mutants, the frzZ mutant was able to respond to attractants and repellents in the spatial chemotaxis assay. The discovery of FrzZ demonstrates that the M. xanthusfrz signal‐transduction pathway utilizes multiple response‐regulator (CheY‐like) proteins.