The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.     

The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521.     

The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development.

The Myxococcus xanthus asgA gene is one of three known genes necessary for the production of extracellular A-signal, a cell density signal required early in fruiting body development. We determined the DNA sequence of asgA. The deduced 385-amino-acid sequence of AsgA was found to contain two domains: one homologous to the receiver domain of response regulators and the other homologous to the transmitter domain of histidine protein kinases. A kanamycin resistance (Kmr) gene was inserted at various positions within or near the asgA gene to determine the null phenotype. Those strains with the Kmr gene inserted upstream or downstream of asgA are able to form fruiting bodies, while strains containing the Kmr gene inserted within asgA fail to develop. The nature and location of the asgA476 mutation were determined. This mutation causes a leucine-to-proline substitution within a conserved stretch of hydrophobic residues in the N-terminal receiver domain. Cells containing the insertion within asgA and cells containing the asgA476 substitution have similar phenotypes with respect to development, colony color, and expression of an asg-dependent gene. An analysis of expression of a translational asgA-lacZ fusion confirms that asgA is expressed during growth and early development. Finally, we propose that AsgA functions within a signal transduction pathway that is required to sense starvation and to respond with the production of extracellular A-signal.     

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus . We report the discovery of four additional pil genes: pilD , a homologue of type IV prepilin leader peptidases; and pilG , pilH and pilI , which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH . Null mutants of pilG , pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.     

Myxococcus xanthus mokA encodes a histidine kinase-response regulator hybrid sensor required for development and osmotic tolerance

A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum.     

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.     

O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis

Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His₁₀-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.     

Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus

Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge.     

Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression

Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Omega4521 fusion are Lac(+). One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of 4521 expression, 11 Lac(-) TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac(+) LPS O-antigen mutants containing Tn5 lac Omega4521 (Tc(r)). Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN. One mapped to sasS, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP. One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in sasP conferred defects during growth and development. These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development.     

MlpA, a lipoprotein required for normal development of Myxococcus xanthus.

The mlpA gene encoding a 236-residue polypeptide has been identified immediately downstream of the oar gene of Myxococcus xanthus (M. Martinez-Canamero, J. Munoz-Dorado, E. Farez-Vidal, M. Inouye, and S. Inouye, J. Bacteriol. 175:4756-4763, 1993). The amino-terminal 21 residues of MlpA encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site. When expressed in Escherichia coli in the presence of [2-3H]glycerol, 3H-labeled MlpA had a molecular mass of 33 kDa and was found to be associated with the membrane fraction. Globomycin, an inhibitor of signal peptidase II, caused a shift in the mobility of E. coli-expressed MlpA to 35 kDa. Subsequently, a mlpA disruption strain (oar+) was constructed and found to have delayed fruiting body formation (by approximately 36 h), with significantly larger fruiting bodies being produced compared with those of the wild-type strain. Nevertheless, spore yields for the two strains were identical after 120 h of development. These data indicate that MlpA, the lipoprotein identified in M. xanthus, is required for normal fruiting body formation.     

RasA is required for Myxococcus xanthus development and social motility

An insertion in the rasA gene entirely blocked developmental aggregation and sporulation in Myxococcus xanthus while also reducing swarm expansion on a 0.3% agar surface. Data presented here demonstrate that rasA is required for extracellular fibril formation and social gliding motility.     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA

The antibiotic TA, a complex macrocyclic polyketide of Myxococcus xanthus, is produced, like many other polyketides, through successive condensations of acetate by a type I polyketide synthase (PKS) mechanism. The chemical structure of this antibiotic and the mechanism by which it is synthesized indicate the need for several post-modification steps, such as a specific hydroxylation at C-20. Previous studies have shown that several genes, essential for TA biosynthesis, are clustered in a region of at least 36kb, which was subsequently cloned and analyzed. In this study, we report the analysis of a DNA fragment, containing a specific cytochrome P-450 hydroxylase, presumably responsible for the sole non-PKS hydroxylation at position C-20. Functional analysis of the cytochrome P-450 hydroxylase gene through specific gene disruption confirms that it is essential for the production of an active TA molecule.     

A Myxococcus xanthus cell density-sensing system required for multicellular development

Abstract Progression through early Myxococcus xanthus multicellular fruiting body development requires the generation of and response to extracellular A signal. Extracellular A signal is a specific set of amino acids at an extracellular concentration greater than 10 μM. It functions as a cell density signal during starvation that allows the cells to sense that a minimal cell density has been reached and development can proceed. The generation of extracellular A signal requires the products of three asg genes. They have recently been identified as AsgA, a fused two-component histidine protein kinase and response regulator; AsgB, a putative DNA-binding protein; and AsgC, the M. xanthus major sigma factor. Other elements of the A signaling pathway map to the sasB locus and appear to be A signal transducers. These elements are regulators of the earliest A signal-dependent gene, whose promoter is a member of the sigma-54 family. Continued study of the A signaling pathway is expected to identify additional components of this network required for the complex behavioural response of fruiting body formation.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.