We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying TnS inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustca, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent.

SUMMARYThe concentration of ribulose 1,5-diphosphate (RuDP) carboxylase, the enzyme which catalyses the conversion of ribulose 1,5-diphosphate + CO, to 3-phosphoglyceric acid, was partially repressed in some Thiorhodaceae organisms when these were grown on certain organic compounds. Transfer of thiosulphate-grown organisms possessing a high concentration of enzyme into growth medium containing pyruvate caused a rapid decline in carboxylase activity. In the reverse situation, pyruvate-grown organisms preferentially synthesized RuDP carboxylase when transferred to growth medium containing thiosulphate alone. The presence of thiosulphate prevented loss of carboxylase with pyruvate. The incorporation pattern of 14C02 into the ethanol-soluble compounds of organisms metabolizing thiosulphate alone was typical of autotrophic metabolism; most of the CO, was fixed via the reductive pentose cycle. The pattern of incorporation of 14C0, by organisms metabolizing pyruvate was strikingly different in that CO, entered the cell constituents predominantly via a carboxylation leading to a four-carbon product. However, even under the latter conditions, the RuDP carboxylase and the reductive pentose cycle appeared to operate to some extent, since phosphoglycerate was an early product of CO, fixation. Phosphoglycerate was an early product of 14C0, fixation by thiosulphate-grown organisms incubated with a variety of organic substrates, showing that the carboxylase and the reductive pentose cycle could function under these conditions. The addition of thiosulphate increased incorporation of 14C0, into phosphate esters by pyruvate-grown organisms incubated with pyruvate. It is concluded that ribulose diphosphate carboxylase and the reductive pentose cycle function in Thiorhodaceae even when grown on organic substrates ; its quantitative importance was not assessed. The synthesis of the carboxylase was influenced by the growth substrate.

A highly polymorphic Xenorhabdus luminescens strain was isolated. The primary form of X. luminescens was luminescent and nonswarming and produced a yellow pigment and antimicrobial substances. The primary form generated a secondary form that had a distinct orange pigmentation, was weakly luminescent, and did not produce antimicrobial substances. Both the primary and secondary forms generated a set of colony variants at frequencies that exceeded normal rates for spontaneous mutation. The variant forms include nonswarming and swarming forms that formed large colonies and a small-colony (SC) form. The primary and secondary forms generated their SC forms at frequencies of between 1 and 14% and 1 and 2%, respectively. The SC forms were distinct from their parental primary and secondary forms in colony and cellular morphology and in protein composition. The cellular morphology and protein patterns of the nonswarming and swarming colony variants were all very similar. The DNA fingerprints of all forms were similar. Each SC-form colony reverted at high frequency to the form from which it was derived. The proportion of parental-type cells in the SC-form colonies varied with age, with young colonies containing as few as 0.0002% parental-type cells. The primaryto-secondary switch was stable, but all the other colony forms were able to switch at high frequencies to the alternative colony phenotypes.

Bacterial isolates of the genus Xenorhabdus were shown to be extremely sensitive to photoproducts produced in a number of common media irradiated by fluorescent light. Two forms of toxic oxygen, hydrogen peroxide and superoxide radical, were produced in the media upon exposure to fluorescent light. The addition of pyruvate or catalase to the irradiated media eliminated the toxicity. The poor plating efficiencies previously reported for Xenorhabdus spp. are likely due to the uncontrolled exposure of media to ambient lighting. This study was prompted by the difficulties we and others have reported in obtaining quantitative data on Xenorhabdus viability because of problems of poor plating efficiency (15, 29, 37). Recently, we have observed that the plating efficiency of several Xenorhabdus strains in our laboratory was greatly reduced on plates exposed to ambient fluorescent light. We report here the extreme sensitivity of Xenorhabdus spp. to a variety of media irradiated with fluorescent light and describe various parameters, including protective agents, of the phenomenon. Bacteria of the genus Xenorhabdus form a complex symbiotic association with the entomogenous nematodes of the families Steinernematidae and Heterorhabditidae (29). In natural conditions, the nematodes and Xenorhabdus act cooperatively to kill insects (9, 10, 15, 29), and each depends on the other for its survival (1, 15, 29). Because a large number of insect species are susceptible to Xenorhabdus-nematode infection, the association has potential for the biological control of insect pests (3). The production of toxic photoproducts in bacterial media by near-UV (NUV) visible light has been reported by a number of workers (4, 6, 16, 18, 19, 31, 35, 39). Although the source, nature, and mechanism of formation of toxic photoproducts produced in these media remain in dispute, H202 is reported to be responsible for much of the toxic activity (4, 6, 14, 16, 19, 26, 31, 39). H202 formation has been shown to occur by the action of visible light on tryptophan (17, 26, 39) and on riboflavin and its derivatives (18, 21). Furthermore, photodynamic action of a variety of biological chromophores, including quinones, pteridines, NAD, thiouracil, pyridoxal phosphate, cytochromes, prophyrins, riboflavin, flavin adenine dinucleotide, and flavoproteins has been shown to produce other oxygen species (e.g., hydroxyl radical [OH-], superoxide radical [02' ], and singlet oxygen [1021) which are toxic to bacteria (14, 21-23, 33). The various Xenorhabdus species and strains used, the culture conditions, and the medium preparation were as previously described (20, 37). All media were kept in the dark except for experimental irradiation. H202 was purchased from Fisher Scientific Co., Pittsburgh, Pa. All other chemicals were purchased from Sigma Chemical Co., St.

Highly purified cell walls of Chromatium vinosum were isolated by differential centrifugation, with or without Triton X-100 extraction. The isolated material had a protein composition similar to that of cell walls obtained by sucrose density gradient centrifugation. Twenty-two proteins were reproducibly detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 42-kilodalton protein was shown to account for 65% of the total cell wall protein. The majority of cell wall proteins were solubilized in sodium dodecyl sulfate at room temperature; however, they existed as high-molecular-weight complexes unless heated to 450C or above. The cell wall contained one heat-modifiable protein which migrated with an apparent molecular weight of 37,400 when solubilized at 700C or below, but which migrated with an apparent molecular weight of 52,500 if solubilized at 1000C. The electrophoretic mobility of three proteins was modified by 2-mercaptoethanol. The majority of C. vinosum cell wall proteins had isoelectric points between pH 4.5 and 5.5, and the 42-kilodalton protein focused at pH 4.9. No proteins were detected which were analogous to the lipoprotein or peptidoglycanassociated proteins of the Enterobacteriaceae. Nearest-neighbor analysis with a reducible, cross-linking reagent indicated that three proteins, including the 42kilodalton protein, associated with themselves. Most of the cell wall proteins were partially accessible to proteases in both intact cells and isolated cell walls. Protease treatment of the whole cell or isolated cell wall digested approximately an 11,000-molecular-weight portion of the 42-kilodalton protein.