Survival and nutritional requirements of three bacteria isolated from ultrapure water

Bacteria isolated previously from ultrapure water (UPW) systems were examined for their ability to survive in UPW, with the ultimate goal of elucidating potential carbon and energy sources for the bacteria. Two strains of Ralstonia pickettii isolated from different areas within the UPW system (pretreatment and polishing loop, and referred to as strains 3A1 and MF254A, respectively) and a strain of Bradyrhizobium sp. were compared to increase our understanding of the fundamental behavior of bacteria contaminating UPW. R. pickettii (3A1) grew significantly slower in R2A medium, with a final cell yield much lower than the isolate from the polishing loop. In addition, R. pickettii MF254A showed a broader substrate range than either strain 3A1 or Bradyrhizobium sp. In UPW, there appears to be a threshold cell concentration (approximately 10⁶ colony-forming units/ml), whereby the cell numbers remain constant for a prolonged period of 6 months or more. Below this concentration, rapid proliferation is observed until the threshold concentration is attained. Preliminary experiments suggested that nitrogen gas (frequently added to UPW storage tanks) may contribute to growth of Bradyrhizobium sp. Above the threshold concentration, the strain of Ralstonia sp. isolated from the polishing loop was capable of cryptic growth with heat-killed cells in UPW. However, cryptic growth was not observed when the cells supplied as nutrients were killed using UV254 light. Furthermore, cryptic growth did not appear to contribute significantly to proliferation of Bradyrhizobium sp. or Ralstonia sp. 3A1 (isolated from the pretreatment loop). We believe that cryptic growth may aid survival of the bacteria in UPW, but further experiments are warranted to prove this phenomenon conclusively. Journal of Industrial Microbiology & Biotechnology (2002) 29, 75–82 doi:10.1038/sj.jim.7000273 Received 23 January 2002/ Accepted in revised form 29 April 2002     

Analysis of bacteria contaminating ultrapure water in industrial systems

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>or=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.     

Case of Localized Recombination in 23S rRNA Genes from Divergent Bradyrhizobium Lineages Associated with Neotropical Legumes

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.     

Genetic Diversity Patterns and Functional Traits of Bradyrhizobium Strains Associated with Pterocarpus officinalis Jacq. in Caribbean Islands and Amazonian Forest (French Guiana)

Pterocarpus officinalis Jacq. is a legume tree native to the Caribbean islands and South America growing as a dominant species in swamp forests. To analyze (i) the genetic diversity and (ii) the symbiotic properties of its associated nitrogen-fixing soil bacteria, root nodules were collected from P. officinalis distributed in 16 forest sites of the Caribbean islands and French Guiana. The sequencing of the 16S-23S ribosomal RNA intergenic spacer region (ITS) showed that all bacteria belonged to the Bradyrhizobium genus. Bacteria isolated from insular zones showed very close sequence homologies with Bradyrhizobium genospecies V belonging to the Bradyrhizobium japonicum super-clade. By contrast, bacteria isolated from continental region displayed a larger genetic diversity and belonged to B. elkanii super-clade. Two strains from Puerto Rico and one from French Guiana were not related to any known sequence and could be defined as a new genospecies. Inoculation experiments did not show any host specificity of the Bradyrhizobium strains tested in terms of infectivity. However, homologous Bradyrhizobium sp. strain-P. officinalis provenance associations were more efficient in terms of nodule production, N acquisition, and growth than heterologous ones. The dominant status of P. officinalis in the islands may explain the lower bacterial diversity compared to that found in the continent where P. officinalis is associated with other leguminous tree species. The specificity in efficiency found between Bradyrhizobium strains and host tree provenances could be due to a coevolution process between both partners and needs to be taken in consideration in the framework of rehabilitation plantation programs.     

Root Nodule Bradyrhizobium spp. Harbor tfdAα and cadA, Homologous with Genes Encoding 2,4-Dichlorophenoxyacetic Acid-Degrading Proteins

The distribution of tfdAα and cadA, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading Bradyrhizobium sp. isolated from pristine environments, was examined by PCR and Southern hybridization in several Bradyrhizobium strains including type strains of Bradyrhizobium japonicum USDA110 and Bradyrhizobium elkanii USDA94, in phylogenetically closely related Agromonas oligotrophica and Rhodopseudomonas palustris, and in 2,4-D-degrading Sphingomonas strains. All strains showed positive signals for tfdAα, and its phylogenetic tree was congruent with that of 16S rRNA genes in α-Proteobacteria, indicating evolution of tfdAα without horizontal gene transfer. The nucleotide sequence identities between tfdAα and canonical tfdA in β- and γ-Proteobacteria were 46 to 57%, and the deduced amino acid sequence of TfdAα revealed conserved residues characteristic of the active site of α-ketoglutarate-dependent dioxygenases. On the other hand, cadA showed limited distribution in 2,4-D-degrading Bradyrhizobium sp. and Sphingomonas sp. and some strains of non-2,4-D-degrading B. elkanii. The cadA genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between Bradyrhizobium sp. and Sphingomonas sp., indicating the incongruency of cadA with 16S rRNA genes. The nucleotide sequence identities between cadA and tftA of 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were 46 to 53%. Although all root nodule Bradyrhizobium strains were unable to degrade 2,4-D, three strains carrying cadA homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of cadA homologs in the cleavage of the aryl ether linkage. Based on codon usage patterns and GC content, it was suggested that the cadA genes of 2,4-D-degrading and nondegrading Bradyrhizobium spp. have different origins and that the genes would be obtained in the former through horizontal gene transfer.     

Diversity and Evolution of Hydrogenase Systems in Rhizobia

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.     

Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.     

Phylogenies of symbiotic genes of Bradyrhizobium symbionts of legumes of economic and environmental importance in Brazil support the definition of the new symbiovars pachyrhizi and sojae

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil (Arachis hypogaea, Acacia auriculiformis, Glycine max, Lespedeza striata, Lupinus albus, Stylosanthes sp. and Vigna unguiculata) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades—B. japonicum and B. elkanii—with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII-recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi, the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium.     

Isolation and Presumptive Identification of Adherent Epithelial Bacteria (“Epimural” Bacteria) from the Ovine Rumen Wall

One hundred sixty-one strains of adherent bacteria were isolated under anaerobic conditions from four sites on the rumen epithelial surface of sheep fed hay or a hay-grain ration. Before isolation of bacteria, rumen tissue was washed six times in an anaerobic dilution solution, and viable bacteria suspended in the washings were counted. Calculation indicated that unattached bacteria would have been removed from the tissue by this procedure, but a slow and progressive release of attached bacteria also occurred. Nevertheless, a wide range of characteristic morphological types remained associated with the epithelium as demonstrated by scanning electron microscopy. Most of these types were represented among the isolates. Characterization and presumptive identification of the isolates showed that 95.0% belonged to previously described genera of functionally significant rumen bacteria, including Butyrivibrio sp. (31.1%), Bacteroides sp. (22.4%), Selenomonas ruminantium (9.9%), Succinivibrio dextrinosolvens (8.7%), Streptococcus bovis (8.1%), Propionibacterium sp. (4.3%), Treponema sp. (3.1%), and Eubacterium sp., Lachnospira multiparus, and Ruminococcus flavefaciens (2.5% each). Eight isolates (5.0%) were not identified. L. multiparus was recovered only from hay-fed animals; all other genera were obtained from animals fed either ration. All S. bovis strains and two strains each of Bacteroides sp. and Butyrivibrio sp. were aerotolerant; all other strains were strictly anaerobic. Bacteria representing the gram-positive, facultatively anaerobic flora associated with rumen wall tissue (R. J. Wallace, K.-J. Cheng, D. Dinsdale, and E. R. Ørskov, Nature (London) 279:424-426, 1979) were therefore not recovered by the techniques used; instead a different fraction of the adherent population was isolated. The term “epimural” is proposed to describe the flora associated with the rumen epithelium.     

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r² = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Distribution and antimicrobial multiresistance in Gram-negative bacteria isolated from Turkish sea bass (Dicentrarchus labrax L., 1781) farm

In this study, the frequencies of 111 antibiotic-resistant Gram-negative bacteria obtained from cultured sea bass were investigated. All the strains were identified and tested whether they were resistive against ten different antibiotics or not. The results showed that most of the bacteria were resistant to trimethoprim-sulphamethoxazole, cephalotin, tetracycline and streptomycin. It was found multiple antibiotic resistance (MAR) index values ranging from 0.3–0.8 for the bacteria isolated from gill. A large number ofPseudomanas putida (25.2%),Moellerella wisconsensis (18%) andPseudomonas fluorescens (10.81%) were identified. Also, strains ofRalstonia pickettii (9%),Vibrio fluvialis (8.1%),Pantoea sp. (7.2%) andErwinia sp. (5.4%) were found. This study suggested that Turkish sea bass farms have antibacterial multiresistance bacteria and may play as a reservoirs response for disease pathogen bacteria.     

Abundance and Diversity of Soybean-Nodulating Rhizobia in Black Soil Are Impacted by Land Use and Crop Management

To investigate the effects of land use and crop management on soybean rhizobial communities, 280 nodule isolates were trapped from 7 fields with different land use and culture histories. Besides the known Bradyrhizobium japonicum, three novel genospecies were isolated from these fields. Grassland (GL) maintained a higher diversity of soybean bradyrhizobia than the other cultivation systems. Two genospecies (Bradyrhizobium spp. I and III) were distributed widely in all treatments, while Bradyrhizobium sp. II was found only in GL treatment. Cultivation with soybeans increased the rhizobial abundance and diversity, except for the soybean monoculture (S-S) treatment. In monoculture systems, soybeans favored Bradyrhizobium sp. I, while maize and wheat favored Bradyrhizobium sp. III. Fertilization decreased the rhizobial diversity indexes but did not change the species composition. The organic carbon (OC) and available phosphorus (AP) contents and pH were the main soil parameters positively correlated with the distribution of Bradyrhizobium spp. I and II and Bradyrhizobium japonicum and negatively correlated with Bradyrhizobium sp. III. These results revealed that different land uses and crop management could not only alter the diversity and abundance of soybean rhizobia, but also change interactions between rhizobia and legume or nonlegume plants, which offered novel information about the biogeography of rhizobia.     

Isolation and characterisation of surface active compound-producing bacteria from hydrocarbon-contaminated environments

Bacteria able to produce surface active compounds (SACs) were isolated from hydrocarbon-contaminated environments. The phylogenetic diversity of the isolates was evaluated by 16S rRNA gene analysis. The production of bioemulsifiers and biosurfactants was determined on strains representative of 18 different bacterial genera. Cupriavidus sp. BSNC28C produced extracellular biosurfactants which reduce the surface tension into the culture medium up to 37.1 mN m⁻¹. Sixteen strains, belonging to 11 different genera, released extracellular emulsifiers able to stabilise oil–water emulsions. Among them, the strains Bradyrhizobium sp. BSNC30A and Bosea sp. BSNC5B showed emulsification activities comparable to those of synthetic surfactants. Overall, the novel SAC-producing strains characterised in this work display promising features for the future development of economically efficient industrial-scale biotechnological processes.     

Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by Streptomyces scabies

Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.     

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.     

Taxonomic characterization of the microorganisms associated with the cultivable diatom Synedra acus from Lake Baikal

Determination of the taxonomic position of microorganisms associated with diatoms was carried out using microscopic, microbiological, and phylogenetic methods. Examination of the cultures of the fresh-water diatom Synedra acus grown in Lake Baikal water by epifluorescence and electron microscopy revealed that bacterial cells colonized the mucilage surrounding the cells of living microalgae and their cell surfaces and penetrate into the frustules of dead diatoms. A total of 13 strains of heterotrophic bacteria were isolated in pure cultures and described. Based on the results of analysis of their morphological, physiological, and biochemical properties, as well as on data obtained by 16S rRNA gene analysis, the following strains were identified to the species level: Sphingomonas sp., Variovorax paradoxus, Pseudomonas fluorescens, Microbacterium trichothecenolyticum, Rhodococcus sp., Caulobacter vibrioides, and Brevundimonas vesicularis.     

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita,important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.