Pasteuria nishizawae sp. nov., a mycelial and endospore-forming bacterium parasitic on cyst nematodes of genera Heterodera and Globodera.

This study describes Pasteuria nishizawae sp. nov., a fourth species of the genus Pasteuria. This mycelial and endospore-forming bacterium parasitizes the adult females of cyst-forming nematodes in the genera Heterodera and Globodera. The distinct ultrastructural features and unique host range found for this bacterium separate it from two closely related species, Pasteuria penetrans, which parasitizes several species of root-knot nematodes of the genus Meloidogyne, and Pasteuria thornei, which appears to parasitize only one species of the root-lesion nematode, Pratylenchus brachyurus. Because these obligate bacterial parasites of nematodes have not been cultured axenically, the taxonomic relationships described here for each species are based mainly on developmental morphology, fine structure of the respective sporangia and endospores, and their pathogenicity on nematode species.

Pasteuria thornei sp. nov. and Pasteuria penetrans sensu stricto emend., mycelial and endospore-forming bacteria parasitic, respectively, on plant-parasitic nematodes of the genera Pratylenchus and Meloidogyne.

Descriptions are presented of two members of the Pasteuria penetrans group of mycelial and endospore-forming bacteria, parasitic on plant-parasitic nematodes. In one case, the epithet P. penetrans sensu stricto emend. has now been limited to members of this group with cup-shaped sporangia and ellipsoidal endospores, parasitic primarily on the root-knot nematode Meloidogyne incognita. The second organism, with rhomboidal sporangia and nearly spherical endospores, which is parasitic primarily on the root-lesion nematode Pratylenchus brachyurus, is assigned to P. thornei sp. nov. An updated and emended description is offered of the genus Pasteuria Metchnikoff 1888 emend. The relationships are analysed among these two nematode parasites and the type species of this genus, P. ramosa Metchnikoff 1888, a parasite of cladoceran invertebrates. Because none of these microbes has been publicly reported to have been cultivated axenically, these relationships are based mainly on morphological, developmental, and pathological criteria.

Formation of halogenated aryl-polyene (xanthomonadin) pigments by the type and other yellow-pigmented strains of Xanthomonas maltophilia.

Based upon visible electronic absorption spectra and mass spectra, yellow-pigmented strains of Xanthomonas maltophilia, including the type strain (ICPB 2648-67 = ATCC 13637) of this species, were shown to produce aryl-polyene (xanthomonadin) pigments. These pigments, which usually occurred in very small quantities, were isolated and studied as isobutyl derivatives. The most common X. maltophilia pigment (Pigment 1), which occurred in 8 of the 12 yellow-pigmented strains examined, was shown to be a monochlorinated aryl-hexaene, molecular ion (M+) 384, with the empirical formula C23H25O3Cl. Pigment 3, M+ 376, which was found as the major pigment in one strain of X. maltophilia and as a minor component in two other strains, probably is the same non-halogenated aryl-heptaene reported previously in Xanthomonas populi and X. juglandis. Although all of these X. maltophilia strains originated from medical rather than phytopathogenic environments, the occurrence of these xanthomonadin pigments in non-phytopathogenic strains emphasizes the chemotaxonomic significance of these aryl-polyene pigments in the genus Xanthomonas.

Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling.

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritin-labeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.

Isolation and characterization of Hfr strains of Erwinia amylovora.

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.

Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity.

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.

Enzymatic degradation of polygalacturonic acid by Yersinia and Klebsiella species in relation to clinical laboratory procedures.

As scored by several specified plating procedures, clinical and environmental strains of Yersinia enterocolitica, Yersinia pseudotuberculosis, and Klebsiella pneumoniae "Oxytocum" showed detectable, albeit generally weak, ability to digest polygalacturonic (pectic) acid. None of these bacterial strains had the vigorous and rapid pectolytic activity on these polygalacturonic acid-containing media that is typical of soft-rot Erwinia species, although some of the Oxytocum strains came fairly close. Analyses of the pectolytic enzyme contents of the cells and culture supernatants of the Yersinia and Klebsiella species revealed that readily detectable quantities of cell-bound polygalacturonic acid trans-eliminase and hydrolytic polygalacturonase were formed by the Yersinia and Klebsiella species; however, the total units of enzyme activity produced by these bacteria were, in general, lower than were produced by soft-rot Erwinia species. Furthermore, unlike the situation in soft-rot Erwinia cultures, these pectolytic enzymes of Yersinia and Klebsiella species were not excreted rapidly and massively into the growth medium. Cultures of other enterobacteria (Citrobacter species, Enterobacter species, Erwinia amylovora, Erwinia herbicola, Escherichia coli, Proteus species, Salmonella typhimurium, and Serratia marcescens) showed no pectolytic ability whatsoever by any of the plating procedures used and (to the extent they were so examined) produced no pectolytic enzymes detectable either in their cells or culture supernatants. This slow or weak release of pectolytic enzymes by Yersinia and Klebsiella species has a bearing on clinical laboratory procedures suitable for detecting their pectolytic activity; methods adequate for this purpose are detailed.

Unusual susceptibility of Erwinia amylovora to antibacterial agents in relation to the barrier function of its cell envelope.

Wild-type strains of the bacterial phytopathogen Erwinia amylovora (the cause of fire blight disease of apples and pears) are markedly susceptible to novobiocin, deoxycholate, and sodium dodecyl (= lauryl) sulfate. The inhibitory concentration, expressed as the concentration causing a 99% inhibition of growth, of these three antibacterial agents were 15 to 100, 40 to 800, and 50 to 800 mug/ml, respectively, depending on the E. amylovora strain. Growth of strains of other Erwinia spp. and Salmonella typhimurium is not affected at all, or is only slightly affected, at these concentrations. Introduction of the F'lac(+), RP1, and R100drd-56 (but not E-lac(+)) plasmids into an E. amylovora strain results in enhanced susceptibility to novobiocin and sodium dodecyl sulfate but not to deoxycholate. E. amylovora wild-type strains spontaneously release a periplasmic enzyme, cyclic phosphodiesterase, but not a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, into the growth medium. Addition of MgCl(2) (20 mM) and NaCl (84 mM) to tryptone broth stimulates the growth of wild-type E. amylovora strains and reduces or eliminates leakage of the periplasmic enzyme. Mutant strains of E. amylovora, selected for resistance to each separate antibacterial agent (or to all three of them), showed a direct correlation (in all but the novobiocin-resistant mutant) between drug resistance and reduced periplasmic leakiness. The relatively low maximum growth temperature (<37 degrees C) of E. amylovora seems unrelated to periplasmic leakage, as judged from the inability of added MgCl(2) to raise the maximum growth temperature, although the generation time at 30 degrees C is reduced from 108 to 54 min upon the addition of 20 mM MgCl(2). The extensive leakage of periplasmic enzyme and unusual drug susceptibility of E. amylovora strains might stem from some defect(s) in some cell envelope component(s) other than the lipopolysaccharide of these bacteria (which contain the usual liposaccharide constituents).

Caprylate-thallous agar medium for selectively isolating Serratia and its utility in the clinical laboratory.

A defined agar medium (hereinafter designated caprylate-thallous [CT5 agar) containing 0.01% yeast extract, 0.1% caprylic (n-octanoic) acid, and 0.025% thallous sulfate is highly selective for all Serratia species and effectively discriminates against most non-Serratia strains likely to be in the same habitats. The selectivity of CT agar is demonstrated by the very high efficiency of colony formation (mean, 80.7% of that on a nonselective complex medium) on CT agar by known Serratia strains and the very low efficiency of colony formation (close to zero) on CT agar by bacterial strains known not to be Serratia. The utility of this medium in actual clinical laboratory practice is demonstrated by the more rapid and higher recovery of Serratia on this selective medium as compared to conventional procedures of in-tandem runs of 513 consecutive urine, feces, and sputum specimens. Pigmented and nonpigmented Serratia strains deliberately added to fecal specimens can be selectively and quantitatively recovered on CT agar. CT agar compares favorably with, or in some cases is an improvement over, other selective media which have been recommended for isolating Serratia. This selective CT agar medium could be quite useful in ecological surveys, especially those related to hospital-acquired infections.

Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D.

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10-15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance, as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.

Gene transmission among strains of Erwinia amylovora.

Stable donor strains of Erwinia amylovora were obtained from strain EA178R(1) (harboring an Escherichia coli F'lac) by selection for clones resistant to curing by acridine orange. These donor strains (EA178R(1)-99 and EA178R(1)-111) transfer chromosomal markers (arg, cys, gua, ilv, met, pro, ser, trp); the frequency of the appearance of recombinants prototrophic for Cys, Gua, Met, Ser, and Trp is highest (> 10(-5)), followed by recombinants prototrophic for Arg, Ilv, and Pro (10(-7) to 10(-5)). The results of interrupted matings, as well as the frequency of transmission of various markers, suggest that cys is transferred as an early marker by both donor strains. The Hfr state of these donor strains is rather likely on the basis of the following observations. The donor strains exhibit a relatively efficient and possibly oriented chromosome transfer; the Lac(+) character is not cured by acridine orange in these donor strains; and these donor strains do not transfer F.