Extracellular Complementation Research Articles

TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli.

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402-410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

Genetic and molecular analysis of cglB, a gene essential for single-cell gliding in Myxococcus xanthus.

Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed DeltacglB mutant were fully restored upon complementation with the cglB wild-type allele. Experiments with a cglB allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate DeltacglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.

VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.

Extracellular complementation and the identification of additional genes involved in aerial mycelium formation in Streptomyces coelicolor.

Morphogenesis in the bacterium Streptomyces coelicolor involves the formation of a lawn of hair-like aerial hyphae on the colony surface that stands up in the air and differentiates into chains of spores. bld mutants are defective in the formation of this aerial mycelium and grow as smooth, hairless colonies. When certain pairs of bld mutants are grown close to one another on rich sporulation medium, they exhibit extracellular complementation such that one mutant restores aerial mycelium formation to the other. The extracellular complementation relationships of most of the previously isolated bld mutants placed them in a hierarchy of extracellular complementation groups. We have screened for further bld mutants with precautions intended to maximize the discovery of additional genes. Most of the 50 newly isolated mutant strains occupy one of three of the previously described positions in the hierarchy, behaving like bldK, bldC, or bldD mutants. We show that the mutations in some of the strains that behave like bldK are bldK alleles but that others fall in a cluster at a position on the chromosome distinct from that of any known bld gene. We name this locus bldL. By introducing cloned genes into the strains that exhibit bldC or bldD-like extracellular complementation phenotypes, we show that most of these strains are likely to contain mutations in genes other than bldC or bldD. These results indicate that the genetic control of aerial mycelium formation is more complex than previously recognized and support the idea that a high proportion of bld genes are directly or indirectly involved in the production of substances that are exchanged between cells during morphological differentiation.

PSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export.

When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

An ABC transporter plays a developmental aggregation role in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.

Methylation of FrzCD defines a discrete step in the developmental program of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.

Role of Agrobacterium virB genes in transfer of T complexes and RSF1010.

Nonpolar virB mutants of Agrobacterium tumefaciens were tested for RSF1010 mobilization and extracellular complementation. virB2 to virB11 were essential for transfer in both assays. virB1 was essential only for high frequency transfer of RSF1010 and VirE2. Coordinated transfer of a preassembled T complex is supported by these data and competition studies.

An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor.

Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium. We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation. Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK-mutant strain extracellularly complement bld261-mutant colonies, and are themselves extracellularly complemented by bldA- and bldH-mutant colonies. The bldK locus, which is located at 5 o'clock on the genetic map and within Asel fragment "N' on the physical map, consists of five adjacent open reading frames. These genes specify homologues of the subunits of the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters. Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer. We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild-type product of the bld261 gene. The BldK-imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action.

Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010

The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.

An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA.

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.

Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development

JD258, a Tn5 insertion mutant of Myxococcus xanthus, was shown to have major defects in three development-associated properties: expression of the developmentally regulated tps gene, spore formation, and production of multicellular fruiting bodies. The defects in tps gene expression and sporulation could be substantially corrected, at the phenotypic level, by mixing JD258 with wild-type cells (extracellular complementation). By this criterion, JD258 appeared to be a new member of a group of conditional developmental mutants that were previously characterized and placed in four extracellular complementation groups (A to D) based on the ability of mutants in one group to stimulate development in mutants belonging to a different group (D. C. Hagen, A. P. Bretscher, and D. Kaiser, Dev. Biol. 64:284-296, 1978). Mutants from groups A, B, C, and D all displayed extracellular complementation activity when mixed with JD258. These results, and other aspects of the phenotype of JD258, indicate that this mutant defines a fifth extracellular complementation group, group E. The M. xanthus esg locus identified by the Tn5 insertion in JD258 was cloned in Escherichia coli and used for further genetic analysis of the locus. These studies indicated that the esg locus resides within a 2.5-kb region of the M. xanthus chromosome and that the locus contains at least two genetic complementation groups. Our results are consistent with a model in which the esg locus controls the production of a previously unrecognized extracellular signal that must be transmitted between cells for the completion of M. xanthus development.

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.

Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor.

The formation of an aerial mycelium by the filamentous bacterium Streptomyces coelicolor is determined in part by a small morphogenetic protein called SapB. A collection of representative bald (bld) mutants, which are blocked in aerial mycelium formation, are all defective in the production of this protein and regain the capacity to undergo morphological differentiation when SapB is supplied exogenously. We now report that most of the bld mutants are rescued for SapB production and aerial mycelium formation when grown near certain other bld mutants. Extracellular complementation experiments of this kind indicate that morphological differentiation is governed by a hierarchical cascade of at least four kinds of intercellular signals. At least one such signal is present in conditioned medium. It is resistant to boiling and protease treatment, and it remains effective even when diluted up to eightfold in fresh medium.

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.

AsgB, a gene required early for developmental signalling, aggregation, and sporulation of Myxococcus xanthus

The asgB genetic locus of Myxococcus xanthus specifies a function which is required early in the developmental pathway leading to aggregation and sporulation in fruiting bodies. The developmental defect of asgB mutants can be compensated by extracellular complementation using either intact wild-type cells or cell-free supernatants conditioned by developing wild-type cells. A Tn5 insertion was isolated closely linked to asgB480 and facilitated the cloning of both the wild-type (asgB+) and the mutant (asgB480) alleles in Escherichia coli plasmid. Tandem duplications of the asgB locus were constructed in M. xanthus; the completely wild-type phenotype of asgB+/asgB480 partial diploids implies that the asgB480 allele is recessive. This finding, along with extracellular complementation by wild-type cells, is consistent with the hypothesis that the asgB+ locus is required to produce a substance with an intercellular signalling function. At least part of the asgB gene was found to lie within a 1.2 kb SmaI DNA fragment. This 1.2 kb fragment, as well as smaller fragments derived from it, were used as DNA probes in RNA/DNA hybrid analyses of transcription in the asgB region. Two small mRNA species were detected, one about 650 bp long, and the other about 500 bp; the two species of mRNAs apparently overlap. Both mRNAs are present in low, but approximately equal amounts, in vegetatively growing cells. This is consistent with the observation that asg mutants display a mutant vegetative phenotype (a change in colony color and spreading behavior) as well as defective development.

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 the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.

Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions.

Certain developmental mutants of Myxococcus xanthus can be complemented extracellularly by wild-type cells. These mutants behave as if they are defective in cell-cell interactions that are required for development. There may be several different interactions because the mutants belong to four extracellular complementation groups (A, B, C, and D). We report here that B- and C- mutations change the pattern of gene expression during Myxococcus development as detected by transcriptional fusions to lacZ mediated by Tn5 lac. The mutant C locus reduced or abolished developmental beta-galactosidase expression from 15 lac fusions that normally begin to be expressed in wild-type cells after 6 hr of development. Expression of these C-dependent lac fusions was restored to C- mutants by adding wild-type cells. The C- mutation did not affect the expression of 10 lac fusions that normally begin to be expressed before 6 hr of development, indicating that the C-mediated cell-cell interaction is required beginning at about 6 hr of development. Cells require the B+ function very early in development because a B- mutation reduced or abolished developmental beta-galactosidase expression from all 26 lac fusions tested, including some that normally begin to be expressed at the onset of development. In a C- mutant and in a B- mutant, some lac fusions responded with reduced beta-galactosidase expression, whereas other fusions, which would normally begin beta-galactosidase expression at about the same time during development, expressed no beta-galactosidase, indicating that developmental genes within a given temporal class display different sensitivities to the absence of cell-cell interactions. Requirements for B+ and C+ function, as well as the previously described A+ function, appear to lie on the same developmental pathway.

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 β-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A − mutations reduced developmental β-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 β-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.