Rhizobium etli USDA9032 engineered to produce a phenazine antibiotic inhibits the growth of fungal pathogens but is impaired in symbiotic performance

Phenazine production was engineered in Rhizobium etli USDA9032 by the introduction of the phz locus of Pseudomonas chlororaphis O6. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish Fix(-) nodules.     

Conserved Symbiotic Plasmid DNA Sequences in the Multireplicon Pangenomic Structure of Rhizobium etli

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.It is becoming clear that bacterial genomes of strains of the same species vary widely both in size and in gene composition (39). An unexpected degree of genomic diversity has been found by comparing whole genomes (39). For instance, in Escherichia coli strains, differences of up to 1,400 kb account for some strain-specific pathogenic traits (5, 56). The extent of intraspecies genome diversity varies in different bacterial lineages. Some species have a wide range of variation; these species include E. coli (42), Streptococcus agalactiae (53), and Haloquadratum walsbyi (34). Other bacteria display only limited gene content diversity; an example is Ureaplasma urealyticum (1, 54). Tettelin and colleagues have suggested that bacterial species can be characterized by the presence of a pangenome consisting of a core genome containing genes present in all strains and a dispensable genome consisting of partially shared and strain-specific genes (53, 54). This concept is rooted in the earlier ideas of Reanney (43) and Campbell (7) concerning the structure of bacterial populations, and it indicates both that there is a pool of accessory genetic information in bacterial species and that strains of the same or even different species can obtain this information by horizontal transfer mechanisms (7, 43).Genome size and diversity are related to bacterial lifestyle. Small genomes are typical of strict pathogens such as Rickettsia prowazekii (2) and endosymbionts such as Buchnera aphidicola (44a). In contrast, free-living bacteria, such as Pseudomonas syringae and Streptomyces coelicolor, have large genomes (4, 6). The bacteria with the largest genomes are common inhabitants of heterogeneous environments, such as soil, where energy sources are limited but diverse (32). An increase in genome size is attributable mainly to expansion of functions such as secondary metabolism, transport of metabolites, and gene regulation. All these features are common to the nitrogen-fixing symbiotic bacteria of legumes, which are collectively known as rhizobia, and their close relative the plant pathogen Agrobacterium. The genomes of such bacterial species have diverse architectures with circular chromosomes that are different sizes or linear chromosomes, like that in Agrobacterium species, and the organisms contain variable numbers of large plasmids (31, 49). Comparative genomic studies have highlighted the conservation of gene content and order among the chromosomes of some species of rhizobia (22, 23, 25, 40). Furthermore, Guerrero and colleagues (25) observed that most essential genes occur in syntenic arrangements and display a higher level of sequence identity than nonsyntenic genes. In contrast, plasmids, including symbiotic plasmids and symbiotic chromosomal islands (like those in Mesorhizobium loti and Bradyrhizobium japonicum) are poorly conserved in terms of both gene content and gene order (21). It is not clear what evolutionary advantage, if any, is provided by multipartite genomes, but some authors have speculated that such genomes may allow further accumulation of genes independent of the chromosome. Recently, Slater and coworkers (46) proposed a model for the origin of secondary chromosomes. Their idea is based on the notion of intragenomic gene transfers that might occur from primary chromosomes to ancestral plasmids of the repABC type. Observations of conservation of clusters of genes in secondary chromosomes or in large plasmids that retain synteny with respect to the main chromosome support this hypothesis (46).We have been studying Rhizobium etli as a multipartite genome model species (23). This organism is a free-living soil bacterium that is able to form nodules and fix nitrogen in the roots of bean plants. The genome of R. etli is partitioned into several replicons, a circular chromosome, and several large plasmids. In the reference strain R. etli CFN42, the genome is composed of a circular chromosome consisting of about 4,381 kb and 6 large plasmids whose total size is 2,148 kb (23). A 371-kb plasmid, termed pSym or the symbiotic plasmid, contains most of the genes required for symbiosis (21). Previous studies have described the high level of genetic diversity among geographically different R. etli isolates (41). The strains are also variable with respect to the number and size of plasmids. Nevertheless, there has been no direct measurement of diversity at the genomic level, nor have comparative studies of shared and particular genomic features of R. etli strains been reported. Therefore, to assess the degrees of genomic difference and genomic similarity in R. etli, we obtained the complete genomic sequence of an additional R. etli strain and partial genomic sequences of six other R. etli strains isolated worldwide. Our results support the concept of a pangenomic structure at the multireplicon level and show that a highly conserved symbiotic plasmid is present in divergent R. etli isolates.     

The Extent of Migration of the Holliday Junction Is a Crucial Factor for Gene Conversion in Rhizobium etli

Gene conversion, defined as the nonreciprocal transfer of DNA, is one result of homologous recombination. Three steps in recombination could give rise to gene conversion: (i) DNA synthesis for repair of the degraded segment, (ii) Holliday junction migration, leading to heteroduplex formation, and (iii) repair of mismatches in the heteroduplex. There are at least three proteins (RuvAB, RecG, and RadA) that participate in the second step. Their roles have been studied for homologous recombination, but evidence of their relative role in gene conversion is lacking. In this work, we showed the effect on gene conversion of mutations in ruvB, recG, and radA in Rhizobium etli, either alone or in combination, using a cointegration strategy previously developed in our laboratory. The results indicate that the RuvAB system is highly efficient for gene conversion, since its absence provokes smaller gene conversion segments than those in the wild type as well as a shift in the preferred position of conversion tracts. The RecG system possesses a dual role for gene conversion. Inactivation of recG leads to longer gene conversion tracts than those in the wild type, indicating that its activity may hinder heteroduplex extension. However, under circumstances where it is the only migration activity present (as in the ruvB radA double mutant), conversion segments can still be seen, indicating that RecG can also promote gene conversion. RadA is the least efficient system in R. etli but is still needed for the production of detectable gene conversion tracts.DNA may be the target of several intracellular and extracellular injuries that can either modify or break it. Many of these may cause, either directly or indirectly, single- or double-strand breaks, leading to replication fork collapses. Independent of their origin, breaks provoke the activation of several pathways that can repair the damage; homologous recombination is the most important of these because of its ability to repair without a loss of information. Besides its role in repair, homologous recombination helps the diversification of the genome through the acquisition of foreign DNA sequences. Paradoxically, recombination also participates in the maintenance of identity among multigenic families, a process known as concerted evolution (38).Concerted evolution can be generated through gene conversion, an outcome of homologous recombination, which is defined as the nonreciprocal transfer of DNA between two or more homologous sequences. In bacteria, this nonreciprocal transfer of information usually entails sizable gene segments (400 to 600 bp are frequent) and occurs at frequencies higher than the mutation frequency (1, 38, 39). The mechanism of gene conversion has as a consequence the spread of sequence polymorphisms present in one of the two recombining homologs; these polymorphisms can be either maintained or eliminated in both sequences, thus giving rise to identity between homologs. The occurrence of gene conversion in bacteria has been either demonstrated experimentally (3, 12, 21, 31; see reference 38 for a review) or inferred from the conservation pattern among repeated genes through phylogenetic analysis (5, 9, 14, 20, 24, 32, 34, 46; see reference 38 for a review).The most accepted model that explains homologous recombination and gene conversion is the double-strand-break repair (DSBR) model (47). This model, characterized for the presence of double Holliday junctions (HJs) (Fig. ​(Fig.1),1), is flexible enough to explain gene conversion and its association with crossovers. In the DSBR model (Fig. ​(Fig.1),1), the length of gene conversion segments can be modulated by three separate events, namely (i) resynthesis of the degraded segment, using information from the uncut homolog (Fig. ​(Fig.1C);1C); (ii) the extent of migration of the HJs (Fig. ​(Fig.1D);1D); and (iii) mismatch repair in the resulting heteroduplex regions (Fig. 1E and F). Consistent with these predictions, inactivation of bacterial systems participating in the generation of the degraded segment, such as the RecBCD or AddAB system (1, 3), or in charge of mismatch repair, such as MutS (1, 39), provokes a reduction in the frequency and/or extent of gene conversion in bacteria. Oddly enough, the extent of migration of the HJs on gene conversion has been a poorly studied factor.Open in a separate windowFIG. 1.Gene conversion under the DSBR model. (A) Two homologous sequences are shown, differing by sequence polymorphisms (black circles). (B) A double-strand break in the recipient homolog is processed (by RecBCD or AddAB) to a gap, generating 3′ tails. (C) After homolog invasion, DNA synthesis (discontinuous lines) fills the gap, and upon ligation, two HJs are formed. At the gap-filling step, gene conversion has occurred because the uncut sequence was the template for gap resynthesis (note the black circles in the cut homolog). (D) HJ migration (by RuvAB, RecG, or RadA) leads to heteroduplex formation. Mismatch repair in the heteroduplex region (mediated by MutS) dictates, depending on the orientation, whether further gene conversion occurs. (E) After HJ resolution, a crossover event with gene conversion to both the gain (black circles in both homologs) and loss of the polymorphisms is generated. (F) Another possible outcome is a crossover event with gene conversion to the gain of both polymorphisms. Only the resolution of HJs leading to crossovers is shown.Movement of the HJs may be a crucial factor to determine the extent of gene conversion, because long heteroduplex regions can be processed afterwards by the mismatch repair system, generating extensive tracts of gene conversion. At least three systems (RuvAB, RecG, and RadA) participate in the migration of HJs in Escherichia coli. RuvA binds to HJs either as a homotetramer (16) or a double homotetramer (35), maintaining the HJs in a planar form; RuvA is also needed for the binding of the RuvB helicase to DNA (33). RuvB forms a hexameric ring responsible for the migration of the HJs away from the initiation site through ATP hydrolysis.The RecG helicase binds to HJs as a monomer; the so-called “wedge” domain in this protein is responsible for both strand separation and processivity (6). RecG was demonstrated in vitro to drive branch migration in the opposite direction of that of RecA (52); consequently, RecG may undo preformed HJs (an antirecombinogenic activity), but it also has recombinogenic activity (30). Although the third protein, RadA, has not been studied in vitro, mutations in the gene displayed genetic synthetic effects with both ruvA and recG mutations (4, 28), indicating that RadA participates in the migration of HJs.Single mutations in ruvB, recG, or radA reduce homologous recombination to about the same extent in Escherichia coli (4, 22), an unexpected result given the differing in vitro activities for RuvB and RecG. The effect of these mutations on gene conversion has been studied only, to our knowledge, in the case of gonococcal pilin variation (a specialized gene conversion system), where mutations in either ruvB or recG equally reduce the frequency of gene conversion (40). As interesting as these data are, there are some doubts as to whether these phenotypes are applicable for all bacteria. For instance, in several bacteria, such as Helicobacter pylori (18, 19), Acinetobacter baylyi (15), and Rhizobium etli (27, 28), inactivation of ruvB reduces recombination markedly, but inactivation of recG enhances recombination. The role of RadA outside of E. coli has been studied only in Bacillus subtilis (7), where it affects chromosome segregation, and in R. etli, where radA mutants were only weakly affected in recombination (28). These data open up the possibility of differing contributions of RuvB, RecG, and RadA to gene conversion in bacteria other than E. coli.One interesting system in this regard is in Rhizobium etli, a symbiotic nitrogen-fixing alphaproteobacterium. R. etli CFN42 has a multipartite genome of 6.53 Mb, harboring approximately 200 reiterated DNA families. More than 133 of these families are comprised of identical repeats longer than 100 bp (13), long enough to be substrates for homologous recombination (43). Sequence identity among members of repeated families in R. etli (at least for the nitrogenase multigene family) is maintained by multiple recombination events, including gene conversion (36). Phylogenetic analysis of the nitrogenase multigene family members in several R. etli isolates is fully consistent with the operation of gene conversion as a homogenizing mechanism (E. Sepúlveda, M. Castellanos, and D. Romero, unpublished results).To gain insight into the mechanism of gene conversion in R. etli, we have studied the anatomies of tracts undergoing gene conversion in this organism (39). Our results revealed that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion tract lengths ranged in size from 150 bp up to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; and (iv) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments (39).The differential roles of RuvB, RecG, and RadA in homologous recombination in this organism were described recently (28). Based on the effects of single and multiple mutations on recombination frequency, we proposed that RuvAB is the main system for migration of HJs, with RadA playing an ancillary role. RecG, in contrast, appears to inhibit recombination, perhaps due to HJ regression. Gene conversion would be an ideal system to test these proposals because it allows us to explore the length of converted segments, a factor highly related to HJ migration. In particular, we predict that in comparison to the wild type, (i) inactivation of radA should not affect gene conversion tract length, (ii) null mutations in recG should lead to longer gene conversion tracts, and (iii) absence of ruvB would instigate a marked reduction in the length of gene conversion segments, or even their disappearance. Evidence reported here fully confirms these predictions.     

A Hydrophobic Mutant of Rhizobium etli Altered in Nodulation Competitiveness and Growth in the Rhizosphere

We isolated and characterized CE3003, a Tn5-induced mutant with altered colony morphology derived from Rhizobium etli CE3. CE3003 produced domed colonies and was highly hydrophobic as indicated by its ability to partition into hexadecane, whereas its parent produced flat colonies and was hydrophilic. On bean plants, CE3003 induced nodules and reduced acetylene. CE3003 and CE3 grew at similar rates when they were grown separately or together in culture medium or inoculated singly onto bean seeds. However, when they were mixed at a 1:1 ratio and applied to seeds, CE3003 achieved significantly lower populations than CE3 in the rhizosphere. Five days after coinoculation of CE3 and CE3003, the population of the mutant was less than 10% of the population of CE3 in the bean rhizosphere. To determine the nodulation competitiveness of the mutant, it was coinoculated with CE3 at various ratios at planting, and the ratio of the nodules occupied by each strain was determined 21 days later. A 17,000-fold excess of CE3003 in mixed inocula was required to obtain equal nodule occupancy by the two strains. A genomic library of strain CE3 was mobilized into CE3003, and we identified a cosmid, pRA3003, that restored the parental colony morphology and hydrophilicity to the mutant. Restoration of the parental colony morphology was accompanied by recovery of the ability to grow competitively in the rhizosphere and to compete for nodulation of beans. The data show an association between cell surface hydrophobicity, nodulation competitiveness, and competitive growth in the rhizosphere in mutant CE3003.     

Characterization of IntA,a Bidirectional Site-Specific Recombinase Required for Conjugative Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42

Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.     

Evolutionary Dynamics of Insertion Sequences in Relation to the Evolutionary Histories of the Chromosome and Symbiotic Plasmid Genes of Rhizobium etli Populations

Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.Insertion sequences (IS) are the smallest transposable elements found in prokaryotes (usually less than 3 kb in size). They encode a transposase and may also encode small hypothetical proteins (4, 9). IS are distinguished by their ability to move within prokaryotic replicons, including both the chromosome and plasmids, and to copy themselves into various genomic sites. In this manner, IS elements can inactivate or alter the expression of adjacent genes (4). When IS occur in two or more identical copies within a genome, they can participate in various types of genetic rearrangements (e.g., duplications, inversions, and deletions), suggesting that IS may play an important role in the evolution of their hosts by promoting genomic plasticity (34). Due to these evolutionary dynamics, the diversity and distribution of IS elements differ greatly between taxa and even within strains of the same species (27).Various theories have been proposed to explain the evolution of IS elements in laboratory model strains and environmental bacterial populations (8, 18, 25, 29). Two main hypotheses seek to explain how these elements are maintained over the long term in their host genomes. The first proposes that they occasionally generate beneficial mutations and therefore may represent a selective advantage to their hosts (34). The second suggests that IS elements are genomic parasites that are maintained by their high rate of transposition and might be disseminated among different bacterial lineages by horizontal gene transfer (HGT). Data supporting the second hypothesis have shown that some IS elements may transpose at high rates upon entering a new host (42). After the initial infection, however, purifying selection may continuously remove these elements from the genome. Thus, IS may undergo an infection-expansion-extinction cycle that allows them to remain in different bacterial populations within the gene pool (42). These two hypotheses are not contradictory, and the evolutionary dynamics and distribution of IS may differ greatly depending on several factors, including (most notably) the rate of transposition and HGT, as well as selective pressures, population size, and the host''s habitat (6, 18, 21, 25, 27, 29).In the nitrogen-fixing symbiotic bacteria of the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium (of the alphaproteobacteria), Cupriavidus, and Burkholderia (of the betaproteobacteria), IS are particularly abundant in symbiotic plasmids (pSym) and symbiotic chromosomal islands (SI) (2, 12, 14, 19, 20, 43). SI and pSym include most of the genes needed to establish symbiosis in the roots of leguminous plants through nodule formation and nitrogen fixation (11). It is generally believed that these elements entered the rhizobial genomes through HGT (39, 40). Comparative genomic analyses have shown that both pSym and SI are highly variable, with the exception of a common set of genes encoding factors critical to nitrogen fixation (nif) and nodulation (nod) (5, 14). SI and pSym have been found to have lower GC contents and different codon usages than the corresponding chromosomal and nonsymbiotic plasmid sequences, suggesting that they were recently acquired by HGT.Some of these symbiotic elements, such as in pSym of Rhizobium etli CFN42 and the SI of Mesorhizobium loti, are conjugative and mobile (30, 32). Genomic analysis of R. etli CFN42 revealed the presence of 39 IS belonging to different families (14); they were found in the chromosome (11 IS); the 371-kb symbiotic plasmid (13 IS); the smaller 192-kb conjugative plasmid, p42a (13 IS); and two other plasmids, p42b and p42c (2 IS). Interestingly, this particular strain shows no evidence of IS disrupting open reading frames (ORFs) or having transpositional activity. However, another 42 incomplete IS may be found in the chromosome, pSym, and the conjugative plasmid; these incomplete sequences are truncated or contain stop codons in their coding sequences.Here, we focused on the dynamics and distribution of IS in different populations of the nitrogen-fixing symbiont R. etli. Since the maintenance of IS in bacterial species might depend on their transpositional activities and horizontal transfer rates, the identification of IS in the same genomic contexts across different strains of the same species could provide new insights into their persistence and divergence over short evolutionary periods. To examine the evolutionary dynamics of IS in natural populations of R. etli, we characterized the distributions, genomic contexts, and sequence variations of IS in isolates of R. etli from three populations with different origins, as well as in some other Rhizobium species. More specifically, we used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 in a collection of 87 strains representing different geographical sites and a gradient of domestication of the bacterial host, the common bean (Phaseolus vulgaris). We also evaluated whether the IS were conserved in the same genomic context relative to their position in R. etli CFN42 and determined the nucleotide sequences of two IS found in most of the isolates. Several population genetic tests applied to these IS, another pSym gene (nodC), and two chromosomal housekeeping genes (dnaB and glyA) suggest that these two IS elements have been inherited vertically and represent recent components of the R. etli gene pool. Finally, the present study strongly suggests that symbiotic plasmids have a recent origin within the R. etli populations.     

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc⁻) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc⁻ phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc⁺ strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.     

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N₂-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K₂HPO₄-0.08% MgSO₄ · 7H₂O-0.02% NaCl-0.001% FeCl₃ · 6H₂O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.     

The Ability of Aneurinibacillus migulanus (Bacillus brevis) To Produce the Antibiotic Gramicidin S Is Correlated with Phenotype Variation

Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation for Aneurinibacillus migulanus (formerly known as Bacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999^T contains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895^T (=ATCC 9999^T), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.     

Chiisanoside Is Not Absorbed but Inhibits Oil Absorption in the Small Intestine of Rodents

Chiisanoside is the main component of Acanthopanax sessiliflorus leaves. Simultaneous administration of chiisanoside resulted in a decrease in the plasma TG level and increase of undigested TG in the intestinal lumen after oil gavage to mice. This suggests that chiisanoside has the potential to prevent obesity as a lipase inhibitor which suppresses fat absorption in vivo.     

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

Microbiomes associated with multicellular organisms influence the disease susceptibility of hosts. The potential exists for such bacteria to protect wildlife from infectious diseases, particularly in the case of the globally distributed and highly virulent fungal pathogen Batrachochytrium dendrobatidis of the global panzootic lineage (B. dendrobatidis GPL), responsible for mass extinctions and population declines of amphibians. B. dendrobatidis GPL exhibits wide genotypic and virulence variation, and the ability of candidate probiotics to restrict growth across B. dendrobatidis isolates has not previously been considered. Here we show that only a small proportion of candidate probiotics exhibited broad-spectrum inhibition across B. dendrobatidis GPL isolates. Moreover, some bacterial genera showed significantly greater inhibition than others, but overall, genus and species were not particularly reliable predictors of inhibitory capabilities. These findings indicate that bacterial consortia are likely to offer a more stable and effective approach to probiotics, particularly if related bacteria are selected from genera with greater antimicrobial capabilities. Together these results highlight a complex interaction between pathogens and host-associated symbiotic bacteria that will require consideration in the development of bacterial probiotics for wildlife conservation. Future efforts to construct protective microbiomes should incorporate bacteria that exhibit broad-spectrum inhibition of B. dendrobatidis GPL isolates.     

Streptococcus thermophilus Is Able To Produce a β-Galactosidase Active during Its Transit in the Digestive Tract of Germ-Free Mice

This work presents data on the application of a bacterial luciferase used to monitor gene expression of Streptococcus thermophilus in the digestive tract. The main result is that the bacterium was able to produce an active β-galactosidase in the digestive tract, although it did not multiply during its transit. This production was enhanced when lactose (the inducer) was added to the diet.     

Effect of Antibiotic Growth Promoters and Anticoccidials on Growth of Clostridium perfringens in the Caeca and on Performance of Broiler Chickens

The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the ceaca and on performance of broiler chickens were tested in 2 experiments. The supplements were fed as single feed additives or in some combinations. No clinical signs or lesions caused by coccidia were observed in any of the studies. All supplements had an antibacterial effect on Cp and improved growth rate significantly. Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials. These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects.     

Synaptic Plasticity in the Hippocampus of a APP/PS1 Mouse Model of Alzheimer's Disease Is Impaired in Old but Not Young Mice

Background

Alzheimer disease (AD) is a neurodegenerative disorder for which there is no cure. We have investigated synaptic plasticity in area CA1 in a novel AD mouse model (APPPS1-21) which expresses the Swedish mutation of APP and the L166P mutation of human PS-1. This model shows initial plaque formation at 2 months in the neocortex and 4 months in the hippocampus and displays β−amyloid-associated pathologies and learning impairments.

Methodology/Principal Findings

We tested long-term potentiation (LTP) and short term potentiation (paired-pulse facilitation, PPF) of synaptic transmission in vivo in area CA1 of the hippocampus. There was no difference in LTP or PPF at 4–5 months of age in APPPS1-21 mice compared to littermate controls. At 6 months of age there was also no difference in LTP but APPPS1-21 mice showed slightly increased PPF (p<0.03). In 8 months old mice, LTP was greatly impaired in APPPS-21 animals (p<0.0001) while PPF was not changed. At 15 months of age, APPPS1-21 mice showed again impaired LTP compared to littermate controls (p<0.005), and PPF was also significantly reduced at 80 ms (p<0.005) and 160 ms (p<0.01) interstimulus interval. Immunohistological analysis showed only modest amyloid deposition in the hippocampus at 4 and 6 months with a robust increase up to 15 months of age.

Conclusions

Our results suggest that increased formation and aggregation of beta amyloid with aging is responsible for the impaired LTP with aging in this mouse model, while the transient increase of PPF at 6 months of age is caused by some other mechanism.     

Structural characterization of the K antigens from Rhizobium fredii USDA257: evidence for a common structural motif, with strain-specific variation, in the capsular polysaccharides of Rhizobium spp.

Rhizobium fredii participates in a nitrogen-fixing symbiosis with soybeans, in a strain-cultivar-specific interaction, and past studies have shown that the cell surface and extracellular polysaccharides of rhizobia function in the infection process that leads to symbiosis. The structural analysis of the capsular polysaccharides (K antigens) from strain USDA257 was performed in this study. The K antigens were extracted from cultured cells with hot phenol-water and purified by size exclusion chromatography. We isolated two structurally distinct K antigens, both containing a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). The polysaccharides were characterized by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry, nuclear magnetic resonance spectrometry, and gas chromatography-mass spectrometry analyses. The primary polysaccharide, which constituted about 60% of the K-antigen preparation, consisted of repeating units of mannose (Man) and Kdo, [-->)3-beta-D-Manp-(1-->5)-beta-D-Kdop-(2-->], and a second polysaccharide consisted of 2-O-MeMan and Kdo, [-->)3-beta-D-2-O-MeManp-(1-->5)-beta-D-Kdop-(2-->]. These structures are similar to yet distinct from those of other strains of R. fredii and R. meliloti, and this finding provides further evidence that the K antigens of rhizobia are strain-specific antigens which are produced within a conserved motif.     

Synthetic Lignin Mineralization by Ceriporiopsis subvermispora Is Inhibited by an Increase in the pH of the Cultures Resulting from Fungal Growth

(sup14)C-synthetic lignin mineralization by the basidiomycete Ceriporiopsis subvermispora occurs at the highest rate (about 30% after 29 days) in liquid cultures containing 1% glucose and a growth-limiting amount (1 mM) of ammonium tartrate. The titers of manganese peroxidase (MnP) and laccase are lower in these cultures than in cultures containing 1% glucose and 10 mM ammonium tartrate, where the extent of lignin mineralization in the same period is only about 15%. The inverse correlation between enzyme activity and lignin mineralization is also observed when ammonium tartrate is replaced by ammonium chloride or Casamino Acids as the source of nitrogen. This phenomenon can be explained by a gradual increase in the pH of the medium that takes place only in the cultures with high nitrogen concentrations. Supporting this finding, when cultures with 1 mM ammonium tartrate were grown at different pHs, (sup14)CO(inf2) evolved more rapidly from those with pH values near the optimum for MnP activity. On the other hand, (sup14)CO(inf2) evolution from cultures containing 1% glucose supplemented with 1 mM ammonium tartrate plus 9 mM sodium tartrate was as low as that from cultures with a high ammonium tartrate concentration. Since the changes in the pH of these cultures were not as pronounced as those in cultures containing high nitrogen concentrations, tartrate itself may also be contributing to limit the extent of lignin mineralization. Considering that pH instability seems to constitute a common feature of fungal cultures, precautions must be taken to avoid underestimation of their ligninolytic efficiencies.