A new species of the genus Amycoluta, Amycolata alni, is proposed for strains which were mostly isolated from root nodules and rhizospheres of alder trees and were formerly assigned to the species Amycoluta uutotrophicu. Strains of this new species could be differentiated from representatives of A. uutotrophica by deoxyribonucleic acid homology data (17 to 29% relatedness), as well as by their ability to grow on salicin and D-gluconate, their failure to produce acid from cellobiose and meso-inositol, their resistance to penicillin and carbenicillin (10 pg/ml), and other characteristics. Phenotypic features and low levels of deoxyribonucleic acid homology distinguish A. alni from two other species of the genus Amycolatu, Amycoluta suturneu and Amycolata hydrocuibonoxydans. The type strain of A. alni is strain 3LS (= VKM Ac-901).Eleven phenotypically similar nocardioform actinomycetes having a type IV cell wall composition and lacking mycolic acids were isolated from nitrogen-fixing root nodules and rhizospheres of two alder species, Alnus glutinosa and Alnus incana (29; L. S. Sharaya, Diss. C. Sci. thesis, Institute of Biochemistry and Physiology of Microorganisms, Pushchino, USSR, 1985). According to the identification keys and species descriptions that were available at that time (3,14,15), these isolates were assigned to the species Nocardia autotrophica (20; Y. B. Malishkaite, Diss. C. Sci. thesis, University of Moscow, Moscow, USSR, 1984). Recently, N . autotrophica was included as the type species in the genus Amycolata, which was proposed, along with the genus Amycolatopsis, to accommodate nocardioform actinomycetes having cell wall chemotype IV and lacking mycolic acids (19). Besides the type strain, strain ATCC 19727, the description of Amycolata autotrophica included 28 strains of various origins assigned to N . autotrophica by Gordon et al. (14). Many physiological characteristics exhibited by these 28 strains are variable or different from those of the type strain (19), thus indicating that the species Amycolata autotrophica may be a heterogeneous taxon.Variations in deoxyribonucleic acid (DNA) guanine-pluscytosine (G+C) ratios (60 to 72 mol%) and DNA homology values (37 to 67%) (24) among five strains from the group studied by Gordon et al. (14) have shown that strains of different genospecies may have been placed in Amycolata autotrophica primarily on the basis of physiological criteria.Recently , genetic heterogeneity among strains assigned to Amycolata autotrophica, including the strains from alder associations, was demonstrated by restriction fragment analysis of total genomic DNAs (10). These data showed that the alder isolates are distantly related to the type strain of Amycolata autotrophica.Because of the obvious differences in genome structure among strains of the same species, as well as the abovementioned physiological variations, we compared strains assigned to Am.ycolata autotrophica more extensively. On the basis of DNA-DNA hybridization data and chemotaxonomic and physiological charac...
Part of the 16S rRNA gene was amplified directly from uncultured endophyte populations within the root nodules of Datisca cannabina and three strains isolated from nodules of Alnus glutinosa (AgKG'84/4), Coriaria nepalensis (Cn3), and D. cannabina (Dc2). Sequence comparison based on 930 nucleotides indicated that the endophyte of D. cannabina nodules belongs to the genus Frankia and is highly related to the endophyte of C. nepalensis root nodules. The relatedness of the endophytes of C. nepalensis and D. cannabina nodules was also reflected by closely related nifH sequences amplified from the nodules. 16S rRNA sequence analysis of the noninfective strains obtained from both D. cannabina (Dc2) and C. nepalensis (Cn3) nodules also revealed the close relationship of these strains to the genus Frankia.
Two cDNAs representing different members (agNt84 and ag164) of a gene family encoding glycine- and histidine-rich proteins have been isolated from cDNA libraries from Alnus glutinosa root nodules. Expression of the corresponding genes could only be detected in nodules. With in situ hybridization, the expression in nodules was found to occur in young, infected cells of the prefixation zone (zone 2). The encoded proteins contain putative signal peptides for targeting to the endomembrane system, sharing sequence similarity with signal peptides from plant glycine-rich proteins, among them nodulin 24, a nodule-specific protein from soybean. This similarity suggests that, analogous to nodulin-24, proteins encoded by agNt84/ag164 may be located at the interface between the host plant membrane and the matrix surrounding the endosymbiont. The 3' untranslated regions of the cDNAs contain unusual poly(AT)n stretches that may play a role in the regulation of RNA stability. The protein encoded by agNt84 cDNA was expressed in Escherichia coli as a fusion with maltose-binding protein, and was shown to have the ability to bind to a nickel-chelating resin, indicating that it may function as a metal-binding protein.
Thirty-nine selected Frankia strains belonging to different genomic species were clustered on the basis of their in vitro susceptibility to 17 antibiotics, pigment production and ability to nodulate plants of the genus Alnus and/or the family Elaeagnaceae, or the family Casuarinaceae. The majority of the strains studied fell into three cluster groups, A, E and C, corresponding to the three host-specificity groups, Alnus, Elaeagnus and Casuarina. Within the groups, eight composite clusters, consisting of a t least two strains, and five single-member clusters were recovered at the 0.74 distance level, in good agreement with levels of genetic relatedness between the strains. In addition, five strains were recovered as single-member clusters not in the cluster groups, four of them representing single-member genospecies and one strain not assigned to any known genospecies. The concordance between the phenotypic clusters and the genospecies described previously shows that the grouping may reflect the taxonomic structure of the genus Frankia. For some clusters, differentiating phenotypic characters were found which may be useful for species definition.
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