Short Communication: Optimization of bioprocess conditions for exopolysaccharide production by Klebsiella oxytoca

Optimization of bioprocess conditions increased exopolysaccharide production by a strain of Klebsiella oxytoca from 6g/l to 15g/l; this corresponded to an increase in medium viscosity from 36cP at 12s–1 to 20,000 cP at 0.6 s–1. A combination of equal proportions of tryptone nitrogen and urea nitrogen proved to be the best nitrogen source. Lactose was shown to be the preferred carbon source. At an optimum temperature of 25°C, a pH of 7 was found to be the best for exopolysaccharide production. The concentration of exopolysaccharide produced on whey, enriched whey, enriched whey permeate and lactose-rich medium was comparable.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Cadmium-Resistance Plasmid Affected Cd+2 Uptake More Than Cd+2 Adsorption in Klebsiella oxytoca

A bacterial strain was isolated from Petra City Wastewater Treatment Plant. This isolate was identified as Klebsiella oxytoca based on 16S rDNA analysis. A single plasmid (> 23 kb) was detected in this strain and transformed into Esherichia coli JM83. The transformed E. coli cells exhibited elevated resistance to cadmium as compared to parental plasmid-free cells. The sodium dodecyl sulfate (SDS)-treated cells showed higher efficiency in plasmid curing than the ethidium bromide–treated cells. The ethidium bromide–cured cells grew only in a 10 μ g/ml Cd^{+ 2} minimal tolerable concentration, whereas the SDS-treated cells had no growth in any of the Cd concentrations tested (2, 5, 10, 20, 30, 40, and 50 ppm). Contrary to the Freundlich model, the Langmuir model gave a good fit to the Cd biosorption data by K. oxytoca cells. Plasmid curing caused 80%, 82%, and 70% inhibition in the Cd biosorption, adsorption, and uptake, respectively. Furthermore, the absence of lysine decarboxylase (LDC) activity in the cured strain strongly implies that the structural gene-encoding LDC in this bacterium is plasmid encoded. After curing of the plasmid, 100% of the antibiotic-resistant loci were observed as chromosomal encoded. All of the results shown above indicated that the Cd resistance is plasmid mediated.     

Assignment of biological functions to specific plasmids in a local isolate ofRhizobium cicer

The genetic basis of symbiosis, mucoid structure and melanin biosynthesis in a local isolate ofRhizobium cicer was investigated. The strain was a very effective symbiont and produced substantial amounts of exopolysaccharides and melanin. It harbored three high-molar-mass plasmids of 55, 80 and 130 kb, respectively. Thirty-one melanin production-negative (Mep⁻) derivatives were obtained through plasmid curing. The plasmid patterns of cured derivatives indicated that the loss of 55 and/or 80 kb plasmids was sufficient for obtaining Mep⁻ phenotype. The specific involvement of 55-kb plasmid in melanin production was confirmed upon re-introduction of this plasmid into one of the Mep⁻ derivatives. Further investigation also indicated that the 130-kb plasmid might be necessary for both mucoidal appearance and symbiotic functions.     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

Plasmid mediated metal and antibiotic resistance in marinePseudomonas

Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marinePseudomonas toEscherichia coli occurred during mixed culture incubation in liquid broth at 10^–4 to 10^–5 ml^–1. However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 intoE. coli competent cells was successful; however, the efficiency of transformation (1.49×10² Hg^r transformants g^–1 pMR1 DNA) was low.E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.     

Biotransformation of cholesterol by Micrococcus species mediated by plasmid DNA

A steroid-biotransforming strain RJ6 was identified as Micrococcus roseus. This bacterium has a 10kb plasmid pMQV10. Curing mediated through cultivation of the culture with a low concentration (200ng/ml) of mitomycin C is described. Loss of cholesterol degradation (chol⁺) and streptomycin resistance (Sm^r) phenotypes as a consequence of the loss of plasmid indicate the extrachromosomal location of these two genes in this strain. An electroporation procedure was developed for transformation of cured strain of Micrococcus (RJC6) by plasmids. Frequency of greater than 10⁵ transformants/g DNA was achieved, which is 100-fold higher than the standard transformation procedure that yielded 5.3×10³ transformants/g DNA in the same strain.     

Penicillin production by mutants ofPenicillium chrysogenum resistant to polyene macrolide antibiotics

Summary Polyene-resistant mutants ofPenicillium chrysogenum Wis. 54–1255 have been obtained by stepwise selection in increasing concentrations of polyene antibiotics. From the parent strain, sensitive to 10 g/ml of polyene antibiotics, mutants resistant to fungimycin (1.3 mg/ml), amphotericin B (0. 5 mg/ml), or nystatin (167 g//ml) were obtained. Their penicillin production is different from that of the parent strain and in particular some of the fungimycin-resistant mutants produce higher levels of penicillin.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.     

Degradation of 4′-dimethylaminoazobenzene-2-carboxylic acid by Pseudomonas stutzeri

4-Dimethylaminoazobenzene-2-carboxylic acid (DMBC) was utilized as a necessary carbon and nitrogen source by Pseudomonas stutzeri IAM 12097. o-Aminobenzoic acid (o-ABA), N,N-dimethyl-p-phenylenediamine (DMPA) and cathecol were identified as intermediates of DMBC degradation. DMBC was degraded at a concentration below 70 mol dm^–3. The ability to utilize DMBC in P. stutzeri was lost spontaneously to some extent. When P. stutzeri was cured of plasmid DNA (approximately 8 MDal) by treatment with mitomycin C, acridine orange, and chloramphenicol, DMBC was not utilized by the resultant strain. These facts suggest that the degradative ability on DMBC in P. stutzeri is controlled by plasmid DNA. Correspondence to: C. Yatome     

Biopolymer production by a Klebsiella oxytoca isolate using whey as fermentation substrate

A strain of Klebsiella oxytoca was isolated from milk capable of completely utilising the lactose (3.5%, w/v) in whey and producing biopolymer. In a broth containing 5% (w/v) lactose, 6.1 g/l of extracellular biopolymer was produced in 72 h by the isolate. At a shear rate of 2 s, broth viscosities of greater than 400 cP were obtained in lactose rich (4%, w/v) media containing 0.2% (w/v) nitrogen concentration.     

Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12

Summary The product of the dye gene of Escherichia coli, mapping at 99–100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins. Mutation of dye thus results in loss of expression of the F-factor (Fex^–, i.e. male sterility, and dye sensitivity (Dye^s). We have isolated a plasmid, pRB38, in which a 6 kb SalI fragment carrying the dye ⁺ gene was cloned into the plasmid pACYC184. This 6 kb SalI fragment also carries two nearby markers, chlG, involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase.Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure. The product of the dye gene was found to be a polypeptide of M_r=29,000. Thus derivatives of pRB38 in which the transposon was inserted into dye, resulting in a Dye^S Fex^– phenotype when these plasmids were in a dye strain, failed to produce this polypeptide and in some cases produced a truncated product. Such insertions also resulted in a Chl^r and Pho^– phenotype when the plasmid was in a (dye-chlG-phoM) phoR strain, although complementation tests suggested that the phoM ⁺ and chlG ⁺ genes were still intact. Insertions of into the promoter distal end of dye did not result in a Dye^S Fex^– phenotype, although a truncated Dye protein was synthesised, and a Chl^r Pho^– phenotype was produced.It has been suggested (Gaffney et al. 1983) that the dye (=sfrA) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane. Our results suggest that the Dye protein is also required for expression of the molybdenum cofactor and of alkaline phosphatase, and could perhaps be involved in the translocation of these proteins to the membrane.     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

紫云英根瘤菌质粒功能研究

紫云英根瘤菌CH203含有3条质粒(pRHa,97MI);pRHb,168MD;pRHc,251MD为共生质粒),用带蔗糖敏感基因Tn5-sacB进行菌株质粒消除和质粒缺失突变株筛选,获得一系列突变株。与野生型菌相比,质粒pRHa的丢失导致菌株结无效根瘤,质粒pRHb的丢失使菌株失去共生能力,在TY培养基平板上菌落变得粗糙,失去了脂多糖(LPSI)。质粒pRHc(共生质粒)的丢失显然失去其菌株的共生能力,同时使菌株抗酸性明显减弱。质粒回复能恢复突变株的表现特征和共生能力。此外,紫云英根瘤菌CH205含有5条大小不同的质粒(分子量42MD～230MD),该菌株某些质粒的消除能显著增强菌株的结瘤固氮能力。研究结果也表明除共生质粒外,紫云英根瘤菌其它质粒明显影响菌株的共生效应。     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

Fungal populations and mycotoxins in silage in Assiut and Sohag governorates in Egypt,with a special reference to characteristic Aspergilli toxins

Forty silage samples were collected from Assiut and Sohag governorates in Egypt to measure the presence of fungal population in silage. Forty-three species and 2 species varieties belonging to 17 genera were isolated using glucose Czapeks and Sabourauds dextrose agar media at 28 ^°C. The most prevalent genera were Aspergillus (57.5 and 100 of the samples), Penicillium (100 and 55%) on the two mentioned media, respectively. Also, Fusarium oxysporum and Gibberella fujikurori were recovered in moderate incidences. Mycotoxin profiles were also determined in these samples: Aflatoxins showed the highest incidence rates of occurrence, it occurred in 22.5% of all samples analyzed. Other mycotoxins were detected from all samples (T₂ toxins and sterigmatocystin at incidence of 7.5 and 5%, respectively). The screening of the characteristics mycotoxins of different isolates of Aspergillus isolated from silage samples was tested. The results clarified that some mycotoxins (aflatoxins – aspergillic acid – beta nitro propionic acid – cyclopiazonic acid– kojic acid and sterigmatocystin) were produced by some isolates of A. flavus. Some isolates of A.fumigatus could produce gliotoxin and verrucologen. All of A. niger isolates tested were able to produce kojic acid. One isolate of A. ochraceous formed ochratoxin A and other isolate produced penicillic acid. Concerning A. terreus isolates, the results showed that 5 isolates were able to produce citrinin and 4 isolates had ability to produce patulin. A. versicolor isolates showed the ability to produce ochratoxin A.     

Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography

We report for the first time the use of liquid-liquid counter-current chromatography (CCC) for the preparative scale fractionation of plasmid DNA. Almost complete fractionation of supercoiled and open circular plasmid DNA (6.9 kb) could be achieved using a phase system comprising 12.5% (w/w) PEG 600 and 18% (w/w) K₂HPO₄. Experiments were carried out on a Brunel J-type CCC machine (100 ml PTFE coil) at a mobile phase flow rate of 0.5 ml min^{– 1} and a rotational speed of 600 rpm. Compared to conventional HPLC techniques the capacity of CCC is not limited by the surface area of resin available for adsorption. Symbols: C _b, Concentration of plasmid in lower phase (g ml^–1); C _t, Concentration of plasmid in upper phase (g ml^–1); CV, Total volume of mobile phase present in the coil and connecting leads (ml); K, Equilibrium solute partition coefficient (K=C _t/C _b); OC, Open circular plasmid; SC, Supercoiled plasmid; S _f, Percentage stationary phase retention (S _f=V _s/V _c); t _s, Time for phase separation (s); V _b, Volume of bottom phase (ml); V _c, Coil volume (ml); V _m, Volume of mobile phase present in coil at equilibrium (ml); V _r, Volume ratio of two phases (V _r=V _t/V _b); V _s, Volume stationary phase present in coil at equilibrium (ml); V _t, Volume of top phase (ml); V _tot, Total volume of phase system (ml).     

An extracellular sulfated fucose-rich polysaccharide produced by a tropical strain of Cryptomonas obovata (Cryptophyceae)

A tropical strain of Cryptomonas obovata Skuja, isolated from a shallow oxbow lake,releaseda sulfated fucose-rich polysaccharide. The polysaccharide is composed mainly offucose (42%), N-acetyl-galactosamine (26%) and rhamnose (15%), with smallquantities of glucuronic acid, mannose, galactose, xylose and glucose. Sulfateaccounted for 1.7% total polysaccharide. Quantitative release was studied withcells exposed to optimal culture conditions contrasted with high irradiance andnitrate depletion. This latter set of conditions could simulate stresssituations usually found in the place from which this strain was isolated. Themonosaccharide composition of the polysaccharide was evaluated using PAD-HPLCand gas chromatography. The two irradiances tested (165 molm^–2 s^–1 and 2000 molm^–2 s^–1) had no significant effect onamounts of polysaccharide released by the cells. Differences were observed whenthe nitrate availability was varied. In the nitrate-depleted situation,extracellular polysaccharide production was 2.5 times higher than replete cellsafter 6 h at 165 mol m^–2s^–1, and 2.25 times higher at 2000 molm^–2 s^–1.