H2-Recycling system in mungbean Rhizobium in relation to N2-fixation

Thirty isolates of mungbean Rhizobium were tested for the presence of H₂-recycling system. All the isolates were preliminary screened for detecting H₂-recycling system in free culture using triphenyltetrazolium chloride reduction as screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle hydrogen both in vivo as well as in vitro. Nitrogen fixing efficiency of hydrogenase positive, hydrogenase negative isolates and Hup^– mutants was compared by green house experiments. There was 13–56% increase in dry matter and 21–46% increase in total nitrogen of the plants inoculated with H₂-recycling isolates over the plants inoculated with non-recycling isolates. There was reduction in dry matter and total nitrogen content of the plants inoculated with Hup^– mutants as compared to plants inoculated with wild type strain. The per cent decrease due to inoculation with Hup^– mutants over wild type strain was 19–22 and 20–26 of dry weight and total nitrogen in plants, respectively.Abbreviations TTC triphenyltetrazolium chloride     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae

Improvement in H₂ production was achieved through redirection of metabolic pathways by blocking formation of alcohol and some organic acids in Enterobacter cloacae IIT-BT 08. The wild type strain was more susceptible to allyl alcohol (7 mM) and to the combined effect of NaBr and NaBrO₃ (40 mM each at pH 5.5) than were double mutants, with defects in both alcohol and organic acid formation pathways, which had higher H₂ yields (3.4 mol mol^–1 glucose) than the wild type strain (2.1 mol mol^–1 glucose).     

Neutral sugars in lipopolysaccharides ofRhizobium trifolii and its non-nodulating mutant

Summary The nodulatingRhizobium trifolii strain 24 and its non-nodulating mutant 24 nod^–3 have been examined. The exopolysaccharides of both cultures studied contained mannose, galactose and glucose at similar molar ratios. On the other hand some quantitative differences have been found between the lipopolysaccharides in respect of the composition of neutral sugars. Glucose and rhamnose were the main constituents of the nodulating strain 24, whereas rhamnose and galactose in non-nodulating mutant 24 nod^–3 deprived of the plasmid pWZ2.     

Clostridium acetobutylicum mutants isolated for resistance to the pyruvate halogen analogs

Pyruvate halogen analogs, 3-fluoropyruvate and 3-bromopyruvate, are toxic towardClostridium acetobutylicum. After mutagenesis with nitrosoguanidine, mutants resistant to these compounds were selected. In a normal batch culture regulated at pH 4.8, mutants quickly initiated the acid-solvent transition, accumulating more acetoin and lactate than the wild type strain. The maximum rate of specific glucose uptake was higher in the mutants than in the wild type: 1.76 h^–1 and 0.9 h^–1 respectively. When the pH was uncontrolled, mutants converted glucose into solvents, essentially butanol, while the wild-type strain ceased its fermentation at the acidogenic stage. Enzymatic investigations revealed that acetate kinase, butyrate kinase, and acetoacetate decarboxylase activities decreased sooner in the mutants than in the parent strain.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

A relationship between RP4 plasmid acquisition and phenotypic changes in Pseudomonas fluorescens R2fN

The physiological behaviour of Pseudomonas fluorescens strain R2fN was compared to that of transconjugants [R2fN(RP4)], and two aggregation phenotypes were identified (Agr^– and Agr⁺). Agr⁺ phenotype is characterized by the appearance of macroscopic aggregates when cells are growing in liquid media. Transconjugants exhibited Agr⁺ phenotype whereas wild type strain represented Agr^–. Evidence is presented to support correlation between Agr⁺ phenotype acquisition and the presence of the broad-host range plasmid RP4 in strain R2fN. In addition, according to bacterial adherence to hydrocarbon test the transconjugant cells appeared to be very hydrophilic whereas wild type R2fN cells were hydrophobic.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach

This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100–1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12–30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 °C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS–PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.     

Energy trapping and detrapping by wild type and mutant reaction centers of purple non-sulfur bacteria

Time-correlated single photon counting was used to study energy trapping and detrapping kinetics at 295 K in Rhodobacter sphaeroides chromatophore membranes containing mutant reaction centers. The mutant reaction centers were expressed in a background strain of Rb. sphaeroides which contained only B880 antenna complexes and no B800-850 antenna complexes. The excited state decay times in the isolated reaction centers from these strains were previously shown to vary by roughly 15-fold, from 3.4 to 52 ps, due to differences in the charge separation rates in the different mutants (Allen and Williams (1995) J Bioenerg Biomembr 27: 275–283). In this study, measurements were also performed on wild type Rhodospirillum rubrum and Rb. sphaeroides B880 antenna-only mutant chromatophores for comparison. The emission kinetics in membranes containing mutant reaction centers was complex. The experimental data were analyzed in terms of a kinetic model that involved fast excitation migration between antenna complexes followed by reversible energy transfer to the reaction center and charge separation. Three emission time constants were identified by fitting the data to a sum of exponential decay components. They were assigned to trapping/quenching of antenna excitations by the reaction center, recombination of the P⁺H^– charge-separated state of the reaction center reforming an emitting state, and emission from uncoupled antenna pigment-protein complexes. The first varied from 60 to 160 ps, depending on the reaction center mutation; the second was 200–300 ps, and the third was about 700 ps. The observed weak linear dependence of the trapping time on the primary charge separation time, together with the known sub-picosecond exciton migration time within the antenna, supports the concept that it is energy transfer from the antenna to the reaction center, rather than charge separation, that limits the overall energy trapping time in wild type chromatophores. The component due to charge recombination reforming the excited state is minor in wild type membranes, but increases substantially in mutants due to the decreasing free energy gap between the states P^* and P⁺H^–.Abbreviations PSU photosynthetic unit - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - P reaction center primary electron donor - RC reaction center - Rb. Rhodobacter - Rs. Rhodospirillum - EDTA (ethylenediamine)tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Author for correspondence     

Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom

We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala¹⁴–Leu¹⁵ and Tyr¹⁶–Leu¹⁷ but it is inactive on fibrin, plasminogen and prothrombin. We used 8–10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn–Asn–Leu–Thr–Arg–Ile–Val–Gly–Gly—factor X fragment was cleaved by enzyme at positions Leu³–Thr⁴ and Arg⁵–Ile⁶. The fibrinogen peptide fragment Glu–Tyr–His–Thr–Glu–Lys–Leu–Val–Thr–Ser was hydrolysed at position Lys⁶–Leu⁷.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group

Summary Two hundred and eleven nitrate reductase-deficient mutants (NR^–) were isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures by chlorate selection and regenerated into plant. More than 40% of these clones were classified as cnx and presumed to be affected in the biosynthesis of the molybdenum cofactor, the remaining clones being classified as nia mutants. A genetic analysis of the regenerated plants confirmed this proportion of nia and cnx clones. All mutants regenerated were found to carry monogenic recessive mutations that impaired growth on nitrate as sole nitrogen source. Mutants propagated by grafting on N. tabacum systematically displayed a chlorotic leaf phenotype. This chlorosis was therefore related to the NR deficiency. The observation of leaves with NR^– chlorotic sectors surrounded by NR⁺ wild-type tissues suggeests that an NR deficiency is not corrected by diffusible factors. Periclinal chimeras between wild-type tobacco and the NR^– graft were also observed. In this type of chimeric tissue chlorosis was no longer detectable when NR⁺ cells were in the secondmost (L2) layer, but was still detectable when NR^– cells were in the secondmost layer. The genetic analysis of nia mutants revealed that they belong to a single complementation group. However three nia mutants were found to complement some of the other nia mutants. The apoenzyme of nitrate reductase was immunologically detected in several nia mutants but not in other members of this complementation group. Some of the nia mutants, although they were NR^–, still displayed methylviologenitrate reductase activity at a high level. These data show that the nia complementation group corresponds to the structural gene of nitrate reductase. Some of the mutations affecting this structural gene result in the overproduction of an inactive nitrate reductase, suggesting a feedback regulation of the level of the apoenzyme in the wild type.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Conjugational transfer of the nodulation-conferring plasmid pWZ2 in Rhizobium trifolii

Summary When the nodulating Rhizobium trifolii strain 24Vio^r containing plasmid RP4 was conjugated with the non-nodulating R. trifolii mutant strain 24Str^rNod^-35, plasmid RP4 was transferred at a frequency 10^-3–10^-4. Two out of nearly three thousand tested transconjugants which contained plasmid RP4 had acquired the ability to form nodules on clovers. Molecular studies of the DNA of both these nodulating transconjugants showed the presence of plasmid RP4 and another plasmid which was not found in the original recipient strain. The size of this second plasmid corresponded to that of the plasmid pWZ2, the elimination of which was correlated with irreversible loss of the nodulating ability of R. trifolii strain 24 (Zurkowski and Lorkiewicz 1979). Plasmid RP4 was eliminated from cells by ethidium bromide, without the loss of nodulating properties. The nodulation capacity, however, was eliminated from transconjugants after incubation of bacteria at elevated temperature. Non-nodulating clones obtained after such incubation did not contain the plasmid pWZ2. The results indicate that the plasmid pWZ2 is a necessary element for induction of nodules by R. trifolii, and that it can be mobilized by plasmid RP4.     

Mitotic segregation in hybrid of methylotrophic yeastCandida pelliculosa

Protoplasts from two autoxotrophic mutants of methylotrophic yeastCandida pelliculosa were fused by the PEG technique, and the genetic structure of the hybrid obtained was studied by means of mitotic segregation. Spontaneous mitotic segregation depended on the composition of the medium and the type of carbon source used. Mechanisms by which segregants appeared were studied by means of UV- and benomyl-induced mitotic segregation. Our data suggest that one large percentage of segregants (Leu ^–) occurred as a result of mitotic chromosome loss. The other large portion of segregants (Ade ^– andLys ^–) are a consequence of mitotic recombination.     

Response to carbon-starvation in Pseudomonas aeruginosa strain IE-6S+: analysis of general cross protection,production of some nematocidal compounds in vitro,and the biological control of Meloidogyne javanica in tomato

Adaptation to nutrient-limited conditions by repeated culture on soil agar media was found to induce resistance to osmotic, oxidation, thermal and pH stress as well as carbon-limited culture conditions in Pseudomonas aeruginosa strain IE-6S⁺. Culture filtrate of the resistant strains obtained from 10% strength King's medium B (KMB) caused greater (32–54%) mortality of Meloidogyne javanica juveniles compared with their parental strain. When 10% strength KMB was amended with 1% (w/v) glucose, the ability to cause nematode mortality was substantially enhanced by adapted strains, while activity of the parental strain was repressed. Two of the four starved bacteria IE-6S⁺PBK1 and IE-6S⁺KUC2 grown in KMB liquid medium amended with glucose synthesized salicylic acid (5.1 and 5.8 g ml^–1, respectively) and hydrogen cyanide (picrate paper turned yellow to brownish red for both strains) in greater quantities compared to wild type strain (SA = 4.4 g ml^–1, picrate paper turned orange-yellow). Neither wild type strain IE-6S⁺ nor its adapted strains were capable of utilizing tomato root exudates as a sole carbon source. Strains adapted to carbon-limiting conditions exhibited enhanced colonization in the rhizosphere and inner root tissues of tomato compared to their exponentially growing counterpart. Pre-adapted bacterial inoculants applied in the soil also caused greater (15%) reduction in nematode penetration compared to the parental strain or controls.