Lucy Seldin

Deoxyribonucleic Acid Homology Among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Other Nitrogen-Fixing Bacillus Strains

Deoxyribonucleic acid homology experiments were performed among representative strains of Bacillus polymyxa, Bacillus macerans, and Bacillus azotofixans and other nitrogen-fixing Bacillus strains identified as B. polymyxa-like. All B. azotofixans strains showed less than 20% homology when B. polymyxa or B. macerans was used as a probe. The range of homology among B. azotofixans strains (strain P3L-5T[T = type strain] was used as the probe) varied from 54 to 100%, indicating that these strains comprise a relatively homogeneous species. Strain Hino appears to be a variant of B. azotofixans because it exhibited only 28 to 44% homology with this species. Strains P3L-2, CR5D-16, and CR5D-23 showed less than 20% homology with B. polymyxa, B. macerans, and B. azotofixans, indicating that these strains do not belong to these species. Additional data will be necessary to fully clarify the taxonomy of these nitrogen-fixing Bacillus strains.

Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes

ABSTRACT The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp.,Arthrobacter sp., and a bacterium of uncertain affiliation.dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonassp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified asR. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8)dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil.

Bacillus azotofixans sp. nov. a Nitrogen-Fixing Species from Brazilian Soils and Grass Roots

A new species, Bacillus azotofixans, is described. This taxon is based upon 16 soil and root-associated strains that exhibit acetylene-reducing ability and nitrogen-fixing Bacillus sp. strain Hino. B. azotofixans is phenotypically similar to Bacillus polymyxa and Bacillus macerans. However, 13 tests (nitrate reduction; production of acid and gas from xylose, arabinose, lactose, ribose, and glycerol; resistance to lysozyme; liquefaction of gelatin; starch hydrolysis; decomposition of casein and pectin; production of dihydroxyacetone; susceptibility to B. polymyxa phages) differentiate it from B. polymyxa, and 12 characteristics (spore position; Voges-Proskauer test; nitrate reduction; production of acid and gas from xylose, arabinose, lactose, ribose, and glycerol; growth at 45°C; hydrolysis of starch; decomposition of pectin; formation of crystalline dextrins) differentiate it from B. macerans. The guanine-plus-cytosine contents of five strains ranged from 47.9 to 52.5 mol%. All strains reduced acetylene much more efficiently than B. polymyxa or B. macerans. In four strains, nitrogen fixation was confirmed by micro-Kjeldahl analysis of acetylene-reducing cultures. Acetylene reduction was not inhibited by nitrate and was not dependent on yeast extract or thiamin plus biotin. The proposed type strain of B. azotofixans is strain P3L-5 (= ATCC 35681).

Plant Growth Promoting Rhizobacteria: Fundamentals and Applications

Plant growth promoting rhizobacteria (PGPR) have gained worldwide importance and acceptance for agricultural benefits. This is due to the emerging demand for dependence diminishing of synthetic chemical products, to the growing necessity of sustainable agriculture within a holistic vision of development and to focalize environmental protection. Scientific researches involve multidisciplinary approaches to understand adaptation of PGPR, effects on plant physiology and growth, induced systemic resistance, biocontrol of plant pathogens, biofertilization, and potential green alternative for plant productivity, viability of coinoculating, plant microorganism interactions, and mechanisms of root colonization. By virtue of their rapid rhizosphere colonization and stimulation of plant growth, there is currently considerable interest in exploiting these rhizosphere bacteria to improve crop production. The main groups of PGPR can be found along with the phyla Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Therefore, the examples coming up next are related to these microorganisms. Although taxonomic affiliation of validated genera containing PGPR strains described in literature is vast, phenotypic and genotypic approaches are now available to characterize these different rhizobacteria. The progress to date in using PGPR in a variety of applications is summarized and discussed here.

Impact of oil contamination and biostimulation on the diversity of indigenous bacterial communities in soil microcosms

The aim of this study was to analyse the effect of oil contamination and biostimulation (soil pH raise, and nitrogen, phosphate and sulphur addition) on the diversity of a bacterial community of an acidic Cambisol under Atlantic Forest. The experiment was based on the enumeration of bacterial populations and hydrocarbon degraders in microcosms through the use of conventional plating techniques and molecular fingerprinting of samples directly from the environment. PCR followed by denaturing gradient gel electrophoresis (DGGE) was used to generate microbial community fingerprints employing 16S rRNA gene as molecular marker. Biostimulation led to increases of soil pH (to 7.0) and of the levels of phosphorus and K, Ca, and Mg. Oil contamination caused an increase in soil organic carbon (170-190% higher than control soil). Total bacterial counts were stable throughout the experiment, while MPN counts of hydrocarbon degraders showed an increase in the biostimulated and oil-contaminated soil samples. Molecular fingerprinting performed with 16S rRNA gene PCR and DGGE analysis revealed stable patterns along the 360 days of experiment, showing little change in oil-contaminated microcosms after 90 days. The DGGE patterns of the biostimulated samples showed severe changes due to decreases in the number of bands as compared to the control samples as from 15 days after addition of nutrients to the soil. Results obtained in the present study indicate that the addition of inorganic compounds to soil in conjunction with oil contamination has a greater impact on the bacterial community than oil contamination only.

Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils of different texture. DNA and rRNA yields from these soils were 15-30 and 0.25-1.0 μg g-1 soil, respectively. Following different purification steps, the rRNA as well as genomic DNA extracts obtained were sufficiently pure for either reverse transcription and polymerase chain reaction (PCR) amplification, or direct PCR amplification. Using a set of universal bacterial primers based on conserved regions of the 16S rRNA sequence, both approaches yielded mixed target molecules for subsequent denaturing gradient gel electrophoresis fingerprinting of soil microbial diversity. The amplified rRNA-based bacterial diversity assessment was compared with diversity assessments based on amplified DNA in one selected soil. Results showed similarities as well as differences between the profiles generated on the basis of rRNA and those based on genomic DNA, which suggested that the bacterial communities defined on the basis of their genomic DNA contained variable amounts of rRNA. Copyright (C) 1998 Elsevier Science B.V.

Paenibacillus brasilensis sp. nov., a novel nitrogen-fixing species isolated from the maize rhizosphere in Brazil.

Sixteen nitrogen-fixing strains isolated from the rhizosphere of maize planted in Cerrado soil, Brazil, which showed morphological and biochemical characteristics similar to the gas-forming Paenibacillus spp., were phenotypically and genetically characterized. Their identification as members of the genus Paenibacillus was confirmed by using specific primers based on the 16S rRNA gene. SDS-PAGE of whole-cell proteins, API 50CH, morphological and biochemical tests, amplified rDNA-restriction analysis (ARDRA), DNA-relatedness analyses, denaturing-gradient gel electrophoresis (DGGE) and 16S rRNA gene sequence determinations were performed to characterize the novel isolates and to compare them to strains of other nitrogen-fixing Paenibacillus spp. Phenotypic analyses showed that the 16 strains were very homogeneous and shared a high level of relatedness with Paenibacillus polymyxa and Paenibacillus peoriae. However, none of the novel isolates was able to ferment glycerol (positive test for P. polymyxa), L-arabinose or D-xylose (positive tests for P. polymyxa and P. peoriae) or utilize succinate (positive test for P. peoriae). Genetic approaches also indicated a high level of similarity among the novel isolates and P. polymyxa and P. peoriae, but the novel strains clearly could not be assigned to either of these two recognized species. On the basis of the features presented in this study, the 16 novel isolates were considered to represent members of a novel species within the genus Paenibacillus, for which the name Paenibacillus brasilensis is proposed. The type strain is PB1 72(T) (= ATCC BAA-413(T) = DSM 14914(T)).

Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi

Aims: To investigate the potential antagonistic activity of Paenibacillus peoriae strain NRRL BD‐62 against phytopathogenic micro‐organisms and to determine the physiological and biochemical characteristics of the antimicrobial compound produced by this strain.

Bacillus nitrogen fixers from Brazilian soils

SummaryFive different Brazilian soils were examined for the incidence of Bacillus nitrogen fixers. These counts were set in relation to the total spore count, and the number of facultative anaerobic spores. Twenty-four Bacillus strains were isolated using media lacking a nitrogen source, identified and their acetylene-reducing ability was determined. Eighteen of these strains were identified asB. polymyxa, and the other 6 were related to this species, but were different in some taxonomical tests. Of the 24 isolated strains, only 13 reduced acetylene; similarly, only 3 out of 9B. polymyxa reference strains were able to reduce acetylene.

Application of a novel Paenibacillus-specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus spp. in the maize rhizosphere.

In this study, a Paenibacillus-specific PCR system, based on the specific primer PAEN515F in combination with bacterial primer R1401, was tested and used to amplify specific fragments of the 16S rRNA gene from rhizosphere DNA. The amplicons were used in a second (semi-nested) PCR for DGGE, in which bacterial primers F968GC and R1401 were used. The resulting products were separated into community fingerprints by DGGE. To assess the reliability of the method, the diversity of Paenibacillus species was evaluated on the basis of DNA extracted directly from the rhizospheres of four different cultivars of maize (Zea mays), i.e. CMS04, CMS11, CMS22 and CMS36, sown in two Brazilian field soils (Cerrado and Várzea). In addition, a clone library was generated from the PCR-generated 16S rDNA fragments, and selected clones were sequenced. The results of the bacterial community analyses showed, at the level of clone libraries, that considerable diversity among Paenibacillus spp. was present. The most dominantly found sequences clustered into 12 groups, each one potentially representing a species complex. Sequences closely affiliated with the P. macerans and P. azotofixans complexes were found in all samples, whereas other sequences were scarcer. Clones affiliated with the latter species complex were most abundant, representing 19% of all clones analysed. The Paenibacillus fingerprints generated via semi-nested PCR followed by DGGE showed a clear distinction between the maize plants grown in Cerrado versus Várzea soils. Thus, soil type, instead of maize cultivar type, was the overriding determinative factor that influenced the community structures of the Paenibacillus communities in the rhizospheres investigated. At a lower level (subcluster), there was a trend for maize cultivars CMS11 and CMS22 on the one hand, and CMS36 and CMS04 on the other hand, to cluster together, indicating that these respective pair of cultivars were similar in their Paenibacillus species composition. This trend was tentatively linked to the growth characteristics of these maize cultivars. These results clearly demonstrated the efficacy of the Paenibacillus-specific PCR-DGGE method in describing Paenibacillus species diversity in rhizosphere soils.