Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants

Summary Symbiotic mutants of Rhizobium meliloti were isolated following Tn5 mutagenesis. Besides four nodulation mutants (Nod^-) unable to induce nodule formation on alfalfa, five infection mutants (Inf^-), which induce the formation of root nodules without detectable infection threads or bacteroids, were obtained. The Inf^- mutants were subdivided into two classes. One class contains mutants which fail to synthesize acidic exopolysaccharide (EPS^-). The other class is comprised of mutants which produce excess amounts of acidic exopolysaccharide (EPS^*). ¹³C nuclear magnetic resonance spectroscopy of the exopolysaccharide isolated from one of the latter type of Inf^- mutant, 101.45, revealed that the side chain of the repeating octosaccharide unit lacks the terminal pyruvate residue. Complementing cosmids were isolated for all Inf^- mutants. In the case of the Inf^- EPS^- mutants the complementing cosmids contain DNA segments which overlap and are part of megaplasmid 2. For two mutants the mutations were found to map on a 7.8 kb EcoRI fragment. In the case of the Inf^- EPS^* mutants the complementing cosmids carry chromosomal DNA. The mutations of two Inf^- EPS^* mutants were localized on a 6.4 kb EcoRI fragment. Coinoculation of alfalfa plants with Nod^- and Inf^- EPS^- mutants resulted in effective symbiosis. The nodules appeared wild type and fixed nitrogen. In constrast, coinoculations with Nod^- mutants and the Inf^- EPS^* mutant 101.45 did not result in the formation of effective nodules.     

Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii

Acetamide, a nitrogen and carbon source for Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wild-type through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm⁺ (utilized acetamide as sole N source) and acm^-. All acm⁺ isolates had acetamidase activity and were obligate phototrophs (i.e. dark-diers). Acm^- isolates had either normal urea assimilation (ure⁺) or lacked all urea pathway activities, namely transport, urea carboxylase and allophanate hydrolase (ure^-). Inheritance patterns for both types indicated single nuclear gene mutations. The acm^- ure⁺ type presumably resulted from a defective acetamidase gene, and the acm^- ure^- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wild-type enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence.Abbreviations F-acetamide fluoroacetamide - F-acetate fluoroacetate - TAP tris-acetate-phosphate medium - CDB Chlamydomonas dilution buffer - TCA trichloroacetic acid - AH allophanate hydrolase - UC urea carboxylase - PAR photosynthetically active radiation - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Genetic control of heterocyst formation in the blue-Green algae Nostoc muscorum and nostoc linckia

Non-heterocystous, non-nitrogenfixing (het ^- nif^-), heterocystous, non-nitrogenfixing (het ⁺ nif^-) and multiple heterocystous, nitrogen-fixing (M-het ⁺ nif⁺) mutants of heterocystous, nitrogen-fixing (het ⁺ nif⁺) wild-type Nostoc muscorum and Nostoc linckia were isolated and characterized with respect to (a) nitrogenfixing activity, (b) reversion frequency, (c) ammonium repressibility of heterocyst formation, (d) heterocyst spacing pattern, and (e) action of L-methionine-DL-sulphoximine (MSO), an inhibitor of glutamine synthetase (GS), on heterocyst regulation. The mutant and revertant results suggest: (i) either involvement of a common genetic determinant in the formation of heterocyst and nitrogenase or the organization of het genes and nif genes in a single operon prone to complete inactivation by a single polar mutation, (ii) non-participation of active nitrogenase in regulation of heterocyst spacing; (iii) involvement of genetic factor(s) in the control of heterocyst spacing pattern in N. linckia, and (iv) apparently different nature of the mechanism of heterocyst inhibition by proheterocyst from that of heterocyst inhibition by NO ₃ ^- or NH ₄ ⁺ . L-Methionine-DL-sulphoximine inhibits growth and causes heterocyst formation in chains in N. linckia growing in nitrogen-free, NO ₃ ^- , NO ₂ ^- or NH ₄ ⁺ medium, thus indicating a close physiological linkage between heterocyst and inorganic nitrogen metabolism regulation.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR^-K⁺) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR⁺K^-SR⁺). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F₁ plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR^-K⁺) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.     

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 μg or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif⁺ plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his⁺ strain, and was also Nif^- on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

Apparent antimutagenic effect of ultraviolet irradiation

Strain SL3367 is a S. typhimurium LT2 hisG46 stock which spontaneously reverts to His⁺ at a high frequency. Plates of defined medium with 1% (v/v) nutrient broth inoculated with ca. 10⁸ washed SL3367 cells were incubated, untreated or after UV irradiation. After 2 days at 37°C, an average of 165 His⁺ colonies were obtained per control plate but significantly fewer, 105 His⁺ colonies, on plates irradiated at a fluence of 7 J/m². The dry weight of bacteria in washings from plates incubated 14 h (by which time growth of His^? cells had ceased) was the same for irradiated and non-irradiated plates but the yield of colony-forming units from irradiated plates was less than from control plates, by about the same factor as the reduction in yield of His⁺ colonies caused by the same fluence. Washings from incubated irradiated plates, but not those from control plates, contained long filaments as well as bacteria of normal size; on transfer to nutrient-agar slide cultures cells normal size grew into microcolonies but filaments did not grow. The reduced plateau yield of viable His^? cells caused by consumption of much of the growth-limiting supply of histidine by irradiated cells growing into non-viable filaments reduces the number of auxotrophic bacteria at risk for spontaneous reversion and so accounts for the apparent antimutagenic effect of UV irradiation. This effect was partly reversed by the presence of d,l-pantoyl lactone in the selection medium, and was also observed for yield of Trp⁺ colonies from trpE8 cultures with a high spontaneous reversion rate. Treatments not inducing cell filamentation did not result in the depression of spontaneous revertants and were detected as being mutagenic. The apparent antimutagenic effect may be expected for reversion of any auxotroph, unless masked by induced revertants and is particularly apparent in an auxotroph which reverts spontaneously at high frequency.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Genetic analysis of Aspergillus nidulans AmdS+ transformants

Summary To correlate the genetic background of the Aspergillus nidulans amdS deletion strain MH1277 with the integrational behaviour of transforming vectors, classical genetic methods were used to construct AmdS^- strains in which whole chromosomes had been exchanged with those of a master strain. Progeny strains were transformed to the AmdS⁺ phenotype with vector p3SR2. From Southern analysis it was concluded that transformants from all constructions contained tandemly repeated, multiple copy inserts of vector DNA as found for MH1277-derived AmdS⁺ transformants.AmdS⁺ transformants of MH1277 were analysed genetically to prove that the transformant phenotype is genome linked and that transformation by integration can take place on various chromosomes. In one case the AmdS⁺ property showed linkage to both chromosomes II and IV, due to a chromosomal translocation. Sexual analysis of two transformants with AmdS⁺ insertions on the same chromosome revealed a considerable instability of the AmdS⁺ phenotype in one of the strains upon selfing. Due to this instability no decisive answer could be given for the degree of linkage between the AmdS⁺ insertions in these transformants.     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3×10³ s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.     

Carbon and nitrogen source effects on basidiomycetes exopolysaccharide production

The capability to synthesize the extracellular polysaccharide (EPS) is widespread among eight mushroom species which accumulated 0.6–2.2 g/1 of EPS in submerged cultivation. Glucose, maltose, and mannitol were the most appropriate carbon sources for biomass and EPS production. Organic nitrogen sources appeared to be the most suitable nitrogen sources for biomass and EPS accumulation. The cultivation process in shake flasks was successfully reproduced in a laboratory fermentor with enhanced EPS production. The highest yield of EPS (3.8–4.0 g/1) was achieved in cultivation of Agaricus nevoi and Inonotus levis.