Characterization of form variants of Xenorhabdus luminescens.

From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed.     

Colonial and Cellular Polymorphism in Xenorhabdus luminescens

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 primary-to-secondary switch was stable, but all the other colony forms were able to switch at high frequencies to the alternative colony phenotypes.     

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.     

Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.

The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.     

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of the luminescent bacterium Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinducer of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S-and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated inherited variants of P. leiognathi.     

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.     

Identification,characterization, and regulation of a cluster of genes involved in carbapenem biosynthesis in Photorhabdus luminescens

The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed.     

Two new bacterial pathogens of Colorado potato beetle Coleoptera: Chrysomelidae)

Other than Bacillus thuringiensis Berliner, few bacteria are lethal to the Colorado potato beetle (Leptinotarsa decemlineata [Say]), a major pest of potatoes and eggplant. Expanded use of biologicals for the control of Colorado potato beetle will improve resistance management, reduce pesticide use, and produce novel compounds for potential use in transgenic plants. Using freeze-dried, rehydrated artificial diet in pellet form to screen bacteria lethal to other insects, we determined that strains of Photorhabdus luminescens killed Colorado potato beetle larvae. The LC50 for second instar larvae of strain HM5-1 was 6.4 +/- 1.87 x 10(7) cells per diet pellet. In an attempt to find additional naturally occurring P. luminescens strains toxic to Colorado potato beetle larvae, we recovered, from soil, bacteria that produced a purple pigment. This bacterial strain, identified as Chromobacterium sp. by 16S ribosomal DNA sequencing, was also toxic to Colorado potato beetle larvae within 3 d. The LC50 for second instar larvae for these bacteria was 2.0 +/- 0.79 x 10(8) cells per diet pellet, while the LC50 was approximately 1 log lower for third instar larvae. P. luminescens appeared to kill by means of a protein toxin that may be similar to the described lepidopteran protein toxins. Based on the heat and acid stability, the toxin or toxins that Chromobacterium sp. produces, while not fully characterized, do not appear to be typical proteins. In both bacteria, the toxins are made after exponential growth ceases.     

Pigmented variants of Rhizobium

Summary Non-infective pigmented variants of Rhizobium have been isolated from several cultures after treatment with antibiotics, heat and U.V. irradiation. These variants tolerated much higher doses of heat, U.V. and certain antibiotics than the wild type strains from which they originated. Some of the pigmented variants produced reverse mutants on subculture which had lost their pigment but recovered their infectivity. These reverse mutants also lost their extreme U.V.-resistance. However, many pigmented mutants were very stable and reverted only after U.V. irradiation.This investigation shows that pigmentation in Rhizobium and loss of ability to form nodules on the roots of leguminous plants are genetically linked and may be pleiotropic effects. So far, more than 10 pigmented variants have been isolated, none of them was infective, while reverse mutants which had lost their pigment always gave rise to nodules.     

Timing of breeding and offspring number covary with plumage colour among Gyrfalcons Falco rusticolus

Plumage colour variation exists among Gyrfalcons throughout their Arctic and sub‐Arctic circumpolar distribution, ranging from white through silver and grey to almost black. Although different colour variants coexist within many populations, a few geographical regions, such as northern Greenland, possess a single variant, suggesting that local environments may influence plumage colour variation. In central‐west Greenland (66.5–67.5°N), where multiple colour variants exist, white male Gyrfalcons fathered significantly earlier clutches than grey males. No significant association was observed between female colour and lay date. However, significantly more offspring were produced by both male and female white Gyrfalcons than by grey variants when controlling for lay date, and silver Gyrfalcons produced an intermediate number of offspring for both sexes. This pattern was further supported by breeding plumage colour pairings. Grey females paired with grey males nested significantly later in the season and produced fewer offspring than those paired with white males, whereas no difference in lay date or offspring number was found between white males paired with white or with grey females. The difference in the number of offspring produced at each nest‐site was also inversely correlated with the distance to the nearest neighbouring nest, and grey males nested in closer proximity to other nests compared with white and silver colour variants. These results suggest that factors associated with territory occupancy and timing of breeding may regulate reproductive success differently between colour variants, with directional selection favouring light‐coloured Gyrfalcons and resulting in earlier lay date and a high frequency of white plumage colour variants in this population. Although gene flow exists between our study population and those further north (>75°N), white Gyrfalcons prevail where the breeding season duration is even shorter, suggesting that nesting chronology in combination with genetic drift may play an important role in influencing plumage colour polymorphism among Gyrfalcon populations.     

Maintenance procedures for the curtailment of genetic instability: Xanthomonas campestris NRRL B-1459.

Characteristics are described of small-colony variants of Xanthomonas campestris NRRL B-1459 which are frequently encountered when routine culture maintenance procedures are employed. In contrast to the parental type, smallcolony variants were shown to be resistant to a number of antibiotics, to acridine orange, and to phage which are virulent for the parent colony type. Sensitivity to ultraviolet radiation was similar in both colony types. A simple method for preservation of viable cells is described. The suitability of the method for providing reproducible inocula free from variant cell types is examined.     

Maintenance procedures for the curtailment of genetic instability: Xanthomonas campestris NRRL B-1459.

Characteristics are described of small-colony variants of Xanthomonas campestris NRRL B-1459 which are frequently encountered when routine culture maintenance procedures are employed. In contrast to the parental type, smallcolony variants were shown to be resistant to a number of antibiotics, to acridine orange, and to phage which are virulent for the parent colony type. Sensitivity to ultraviolet radiation was similar in both colony types. A simple method for preservation of viable cells is described. The suitability of the method for providing reproducible inocula free from variant cell types is examined.     

Study of the structure of colonies of active and inactive Actinomyces parvullus variants using luminescence and scanning microscopy]

The colony structure of the active and inactive proactinomycete-like variants of Actinomyces parvullus producing actinomycin D was studied with luminescent and scanning microscopy. Clear differentiation of the colony profile was shown by the structure and functions of the mycelium layers. A zone of active synthesis and accumulation of the antibiotic was observed in the colonies of the active variant in the upper part of the substrate mycelium with reddish-yellow self luminescence in UV light and characteristic close hyphae "cemented" by the intracellular substance. Formations of the granule type were often noted on the hyphae of that layer. The layer of the aerial mycelium was loosely connected with the substrate mycelium and consisted of sporophores and spore chains partially broken into single spores. The colonies of the inactive proactinomycete-like variant had a slightly differentiated profile with a sponge-like structure, no zones of the antibiotic synthesis being found. The presence of the intracellular substance was observed in the upper part of the colony supersubstrate mycelium.     

Phenotypic diversification and adaptation of Serratia marcescens MG1 biofilm-derived morphotypes

We report here the characterization of dispersal variants from microcolony-type biofilms of Serratia marcescens MG1. Biofilm formation proceeds through a reproducible process of attachment, aggregation, microcolony development, hollow colony formation, and dispersal. From the time when hollow colonies were observed in flow cell biofilms after 3 to 4 days, at least six different morphological colony variants were consistently isolated from the biofilm effluent. The timing and pattern of variant formation were found to follow a predictable sequence, where some variants, such as a smooth variant with a sticky colony texture (SSV), could be consistently isolated at the time when mature hollow colonies were observed, whereas a variant that produced copious amounts of capsular polysaccharide (SUMV) was always isolated at late stages of biofilm development and coincided with cell death and biofilm dispersal or sloughing. The morphological variants differed extensively from the wild type in attachment, biofilm formation, and cell ultrastructure properties. For example, SSV formed two- to threefold more biofilm biomass than the wild type in batch biofilm assays, despite having a similar growth rate and attachment capacity. Interestingly, the SUMV, and no other variants, was readily isolated from an established SSV biofilm, indicating that the SUMV is a second-generation genetic variant derived from SSV. Planktonic cultures showed significantly lower frequencies of variant formation than the biofilms (5.05 x 10(-8) versus 4.83 x 10(-6), respectively), suggesting that there is strong, diversifying selection occurring within biofilms and that biofilm dispersal involves phenotypic radiation with divergent phenotypes.     

Stability of entomopathogenic bacteria, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>, during in vitro culture

The entomopathogenic nematode–bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.     

Mutations in the csgD promoter associated with variations in curli expression in certain strains of Escherichia coli O157:H7

Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.