The tropical perennial clover, T. semipilosum, and the species, T. repens, exhibited both similarities and differences in their nodulation characteristics following Rhizobium inoculation. Both T. semipilosum and T. repens were infected via root hairs, formed indeterminate nodules and commenced active nitrogen-fixation approximately 1 week after inoculation. Effective Rhizobium strains produced on average four nitrogen-fixing nodules per T. semipilosum plant, predominantly in the upper region of the tap root. In contrast, 65% of the average 15 nodules per T. repens plant were induced on lateral roots. The Rhizobium strain-ANU843, effective (Nod+Fix+) on T. repens, caused root hair branching and twisting on T. semipilosum, although plants did not fix nitrogen, indicative of an ineffective host–strain interaction. Such ineffective interactions were associated with increased total numbers of nodule-like structures on T. semipilosum; these nodules were atypically associated with, or in close proximity to, lateral roots. Using a GUS gene-modified rhizobia to monitor nodule occupancy, a third of the total nodule-like structures formed from the incompatible interactions showed a positive GUS reaction, indicating that colonization by Rhizobium strains of some of the nodule-like structures had occurred. Moreover, about 70% of the GUS positive nodule-like structures from the incompatible interactions were localized at the junction between the tap and lateral roots. These observations are consistent with an atypical infection route by the incompatible strains.

Clostridium acetobutylicum is an anaerobic, motile, industrially important organism. In order to investigate the relationship between motility and solvent production, the gene which encodes the flagellin was cloned, sequenced and the corresponding amino acid sequence deduced. The gene was designated flaC, based upon significant amino acid sequence similarity with flagellin proteins from other organisms. The flaC gene was found to be 825 bp in length which could potentially encode a 275-amino acid protein, with a calculated molecular weight of 29 505Da. Primer extension analysis revealed the presence of a single transcriptional start site 72 bp upstream of the flaC translation start codon, with an inverted repeat present downstream of the structural gene that has the characteristics of a stem-loop structure and thus may act as a rho-independent transcription terminator. The putative promoter that was identified shared strong similarity toσ28 -type promoters that have been identified upstream of flagellin genes in other species. Previous work using western immunoblots had identified that the flagellin protein was a ca. 42-kDa protein, yet this did not agree with the size expected based on nucleotide sequence analysis. PCR analysis revealed that there was no re-arrangement of the flaC structural gene. To further characterize the difference between the two observations, FlaC was investigated for a post-transnational modification such as glycosylation. Protein analysis revealed that the FlaC protein was glycosylated, with a terminal sialyl residue as demonstrated by treatment with neuraminidase.

The ability of Bacillus spp. to alter the nodulation of Phaseolus vulgaris by Rhizobium etli was assessed. The simultaneous presence of both Rhizobium etli TAL 182 and Bacillus megaterium S49 on plant roots during the early stages of plant growth was necessary for enhanced nodulation of Phaseolus vulgaris by the Rhizobium microsymbiont. Coinoculation with both bacterial species also facilitated heterologous nodulation of Rhizobium TAL 182 on Phaseolus acutifolius. These results are consistent with earlier reports of increased root hair proliferation and lateral root formation in response to coinoculation. Split-root experiments revealed that coinoculation partially suppressed host-controlled regulation of nodulation, implicating a plant interaction with the two bacterial species. Changes to the nodulation potential of R. etli due to coinoculation with Bacillus spp. demonstrate the potential for root-associated organisms other than rhizobia to alter the dynamics of the legume–Rhizobium symbiosis.Key words: Bacillus, nodulation enhancement, heterologous nodulation.

A hypervariable region of Rhizobium 23S rDNA was amplified by polymerase chain reaction and phylogenetic relationships of several strains were determined by comparing nucleotide sequences of the amplified product. Variation in the 23S rDNA nucleotide sequences was consistent with phylogenetic relationships determined by host nodulation specificity and (or) 16S rDNA sequence analysis. Six strains representing three Rhizobium species (R. leguminosarum bv. trifolii, R. meliloti, and R. etli), and two strains each of Bradyrhizobium and Agrobacterium were clustered into five rDNA groups. Unique features identified by secondary structure analysis of the 23S rRNA sequenced region were consistent with the hypothesis that 23S rDNA could be used to design species- or strain-specific Rhizobium probes.

Treatment of Phaseolus vulgaris seedlings with the biosynthesis inhibitor, aminoethoxy vinyl glycine (AVG), resulted in a loss of root hair formation. Addition of the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the growth medium enhanced root hair formation in P. vulgaris, in the absence of AVG. The inhibitory effect of AVG on root hair formation was reversed by the combined presence of Rhizobium etli TAL 182 and Bacillus megaterium S49 on P. vulgaris roots. Plants that received single inoculation of either Rhizobium TAL 182 or Bacillus S49 did not show enhancement of root hair formation when AVG was present in the growing medium. These results are consistent with the action of ACC as a positive regulator of root hair morphogenesis. The data suggest that the bacterial interaction may act through the ethylene biosyntheses pathway to enhance root hair morphogenesis, and to disrupt the normal pattern of root epidermal cell development.

Microbial competition for carbon sources is a primary determinant of rhizosphere ecology. We employed the PCR to examine the population fluctuations of a symbiotic nitrogen-fixing bacterium (Rhizobium etli) during the first 11 days following inoculation of Phaseolus vulgaris seedlings grown in the presence or absence of a common asymbiotic rhizosphere resident (Bacillus polymyxa). When B. polymyxa was applied as a co-inoculant, increases in both early rhizobial root populations and final root population densities were observed as compared to single inoculation with R. etli. Modifications to host plant growth (including increased lateral root formation and nodules number) were found concomitant with elevations in R. etli populations on plants co-inoculated with both bacterial genera. In contrast to the in planta results, population enhancements were not observed when R. etli and B. polymyxa were co-cultured in vitro using minimal media in the absence of the seedling. Addition of seed exudate to the growth media also failed to stimulate the population increases observed during co-release in planta. These results suggest that B. polymyxa acts indirectly (i.e., via the plant host) to increase R. etli populations. Our observed synergism among co-resident bacteria supports the hypothesis that microbial communities which colonize the spermosphere may play a significant role in plant development and rhizosphere ecology.

Twenty-two Bacillus spp. isolates from the rhizosphere of Phaseolus vulgaris 'Contender' were identified using Biolog™, gas chromatographic fatty acid methyl ester, and 23S rDNA analyses. Some of the Bacillus isolates produced significant amounts of the phytohormone indoleacetic acid (IAA) when grown in a liquid culture medium supplemented with 100 μg L-tryptophan/L; less IAA was produced in culture medium not supplemented with L-tryptophan. Thin-layer chromatography, high-performance liquid chromatography, gas chromatography – mass spectrometry, and the avena coleoptile bioassay were used to identify and quantify IAA produced by Bacillus isolates. Significant differences were observed in the amounts of IAA produced by different strains of Bacillus, with amounts varying from 0.40 to 4.88 μg/mL. α-Methyltryptophan-resistant mutants of Bacillus exhibited altered IAA production and excreted tryptophan into the growing medium. The IAA-producing Bacillus isolates promoted root growth and (or) nodulation when coinoculated with Rhizobium etli (TAL 182) on Phaseolus vulgaris 'Contender' under gnotobiotic conditions in growth chambers. Coinoculation resulted in increased nodule number, nodule fresh weight, nitrogenase activity, leghemoglobin content, and total soluble protein content in the root nodules of Phaseolus vulgaris. In contrast, coinoculation with α-methyltryptophan mutants resulted in decreased nodulation, indicating that Bacillus isolates have a direct effect on either the Rhizobium or the plant and the effect may not be singularly attributed to their ability to produce IAA in vitro.Key words: Bacillus, indoleacetic acid production, nodulation enhancement.

Pine and spruce seeds were inoculated with antibiotic-resistant plant growth promoting fluorescent Pseudomonas strains Sm3-RN, Ss2-RN and Sw5-RN for evaluation of bacterial root colonization and seedling growth responses under greenhouse conditions. Mycorrhizal inoculum was introduced to seedling containers by placing 2 cc of forest floor soil around seeds at the time of sowing. Mycorrhizal roots were detected on 39% of pine and 30% of spruce seedlings treated with forest soil 13–15 weeks later. Most mycorrhizae were formed by Wilcoxina sp. (E-strain) (89% for spruce and 69% for pine); some Amphinema-like, Mycelium radicis atrovirens, Suillus-like, Thelephora-like, and Tuber-like mycorrhizae were also detected. In the absence of bacterial inoculum, spruce seedling biomass was positively correlated with the number of mycorrhizal root tips. This trend was obscured following inoculation with strains Ss2-RN and Sw5-RN. Pine seedling growth was weakly correlated with mycorrhizal root tip development only after treatment with strain Sm3-RN. Bacterial inoculation did not influence the mycorrhizal status of seedlings, but all three Pseudomonas strains stimulated biomass accumulation of spruce and pine seedlings, up to 19% (P < 0.05). Non-mycorrhizal seedlings tended to support smaller root-associated Pseudomonas populations, but inoculant bacteria colonized seedlings with a minimum of 6.3 × 103 colony forming units per gram of rhizosphere soil regardless of their mycorrhizal status. For spruce, growth effects due to bacterial inoculation were similar in both mycorrhizal and non-mycorrhizal seedlings. However, statistically significant gains in pine biomass by inoculation with strains Sm3-RN and Ss2-RN occurred only in mycorrhizal seedlings, whereas strain Sw5-RN caused significant growth promotion only in non-mycorrhizal pine. Our results suggest that these fluorescent pseudomonad strains enhanced spruce seedling growth through mechanisms unrelated to increased mycorrhizal colonization, but growth promotion of pine by strains Sm3-RN and Ss2-RN was facilitated by an interaction with mycorrhizae.

Microbial competition for carbon sources is a primary determinant of rhizosphere ecology. We employed the PCR to examine the population fluctuations of a symbiotic nitrogen-fixing bacterium (Rhizobium etli) during the first 11 days following inoculation of Phaseolus vulgaris seedlings grown in the presence or absence of a common asymbiotic rhizosphere resident (Bacillus polymyxa). When B. polymyxa was applied as a co-inoculant, increases in both early rhizobial root populations and final root population densities were observed as compared to single inoculation with R. etli. Modifications to host plant growth (including increased lateral root formation and nodules number) were found concomitant with elevations in R. etli populations on plants co-inoculated with both bacterial genera. In contrast to the in planta results, population enhancements were not observed when R. etli and B. polymyxa were co-cultured in vitro using minimal media in the absence of the seedling. Addition of seed exudate to the growth media also failed to stimulate the population increases observed during co-release in planta. These results suggest that B. polymyxa acts indirectly (i.e., via the plant host) to increase R. etli populations. Our observed synergism among co-resident bacteria supports the hypothesis that microbial communities which colonize the spermosphere may play a significant role in plant development and rhizosphere ecology.

A polymerase chain reaction amplification of 23S rDNA was developed to identify Bacillus spp. recovered from roots, mycorrhizae, and rhizosphere soil of conifers. The polymerase chain reaction incorporated a conserved 23S rDNA forward primer in combination with a reverse primer designed to hybridize exclusively to nucleotide sequences of either B. polymyxa or B. mycoides. The amplification provided a rapid and simple means of identifying DNA from isolates of Bacillus, and could be used directly on whole Bacillus cells or mixed populations. The reaction was used to detect and differentiate these Gram-positive species from agar plates inoculated with samples from various conifer samples. A strain-specific primer was also synthesized and used to identify Bacillus which were established within conifer roots 4 weeks after inoculation.

The surface of bacteria consists of polysaccharides, like the exopolysaccharides and lipopolysaccharides and, as it is the case for Azospirillum brasilense, glycoproteins like glycosylated flagella. The precursors for the synthesis of polysaccharides are sugarnucleotides. Several genes, coding for enzymes involved in the synthesis and in the modification of sugarnucleotides, have been characterised is Azospirillum brasilense Sp7.

Thiolase (Thl) is an important enzyme at the junction in the pathway leading to the production of either acids (acetate or butyrate) or solvents (acetone, butanol or ethanol) during the growth of Clostridium acetobutylicum ATCC 824. Cloning and expression of the Thl-encoding gene (thl) has been described [Petersen and Bennett, Appl. Environ. Microbiol. 57 (1991) 2735-2741], as has the purification and properties of the enzyme [Wiesenborn et al., Appl. Environ. Microbiol, 54 (1988) 2717-2722]. Here, we present the complete nucleotide sequence (1.9 kb) of thl. The gene encodes a protein of 392 amino acids (aa) (41237 Da), which mass is in agreement with previous findings, using the purified protein. Primer extension analysis has defined the promoter region, and a stem-loop structure found at the end of thl indicates that it is not part of an operon. The aa sequence of Thl showed homology to those of four other beta-ketothiolases: (i) PhbC of Alcaligenes eutrophus, (ii) PhbA of Chromatium vinosum, (iii) PhbA of Thiocystis violacea and (iv) PhbA of Zoogloea Ramigera. The C terminus of an open reading frame found upstream from the Thl sequence is similar to OrfX of Bacillus subtilis and to NfrC of Escherichia coli.

Annals of the New York Academy of SciencesVolume 721, Issue 1 p. 54-68 Genetic and Metabolic Engineering of Clostridium acetobutylicum ATCC 824a LEE D. MERMELSTEIN, LEE D. MERMELSTEIN Department of Chemical EngineeringSearch for more papers by this authorNEIL E. WELKER, NEIL E. WELKER Department of Biochemistry, Molecular Biology and Cell Biology Northwestern University Evanston, Illinois 60208Search for more papers by this authorDANIEL J. PETERSEN, DANIEL J. PETERSEN Department of Biochemistry and Cell Biology Rice University Houston, Texas 77251–1892Search for more papers by this authorGEORGE N. BENNETT, GEORGE N. BENNETT Department of Chemical EngineeringSearch for more papers by this authorELEFTHERIOS T. PAPOUTSAKIS, Corresponding Author ELEFTHERIOS T. PAPOUTSAKIS Department of Chemical EngineeringTo whom correspondence should be addressed.Search for more papers by this author LEE D. MERMELSTEIN, LEE D. MERMELSTEIN Department of Chemical EngineeringSearch for more papers by this authorNEIL E. WELKER, NEIL E. WELKER Department of Biochemistry, Molecular Biology and Cell Biology Northwestern University Evanston, Illinois 60208Search for more papers by this authorDANIEL J. PETERSEN, DANIEL J. PETERSEN Department of Biochemistry and Cell Biology Rice University Houston, Texas 77251–1892Search for more papers by this authorGEORGE N. BENNETT, GEORGE N. BENNETT Department of Chemical EngineeringSearch for more papers by this authorELEFTHERIOS T. PAPOUTSAKIS, Corresponding Author ELEFTHERIOS T. PAPOUTSAKIS Department of Chemical EngineeringTo whom correspondence should be addressed.Search for more papers by this author First published: June 1994 https://doi.org/10.1111/j.1749-6632.1994.tb47376.xCitations: 21 a This work was supported by National Science Foundation Grants BCS-8912209, BCS-8912094, BCS-9210108, and INT-92102201 (DJP). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume721, Issue1Recombinant DNA Technology IIJune 1994Pages 54-68 RelatedInformation

The ability to genetically alter the product‐formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the “ace operon”) in which the three homologous acetone‐formation genas ( adc , ctfA , and ctfB ) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA‐transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid‐free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22‐fold lower mass of residual carboxylic acids. At all pH values, the acetone‐formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid‐control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid‐borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA‐transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid‐control strain. © 1993 John Wiley & Sons, Inc.

Azospirillum brasilense SP7 cosmid clones which complemented binding of the fluorescent dye calcofluor by Rhizobium meliloti 7027 exoC mutants were identified. One class of cosmids was also found capable of restoring wild-type exopolysaccharide production and beta-1,2-glucan synthesis. A restriction endonuclease map of the A. brasilense DNA insert from complementing clones demonstrated no homology with a previously identified exoC locus. The new (exoC2) locus was further mapped to the A. brasilense chromosome.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.

Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism. The purification and properties of the enzyme have already been described (D. P. Wiesenborn, F. B. Rudolph, and E.T. Papoutsakis, Appl. Environ. Microbiol. 54:2717-2722, 1988). The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19. Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.