Improved electrotransformation frequencies of Corynebacterium

540growth of C.glutamicum strain AS019in glycine or glycine plus INH significantly improved electrotrans-formation efficiency(Haynes&Britz1990).Further-more,we found that a series of mutants,isolated orig-inally as amino acid auxotrophs,protoplasted more readily than their parent,ATCC13059(Best&Britz 1986).These mutants showed significantly different mycolic acid composition and the proportion of extra-cellular mycolic acids was greater following growth in high concentrations of glycine,INH or glycine plus INH(Jang et al.1997).The objective of the present work was to evaluate the use of these cell-surface mu-tants of C.glutamicum(MLB133and MLB194)as recipients in electrotransformation of plasmid DNA and compare these strains with the restriction deficient mutants,RM3and RM4,which are transformed effi-ciently by homologously-and heterologously-derived DNA(Schäfer et al.1994).Materials and methodsBacterial strains,plasmid,and growth conditionsSix strains of C.glutamicum were used:AS019, a spontaneous rifampicin-resistant strain of ATCC 13059(Yoshihama et al.1985);MLB133and MLB194,auxotrophic mutants(ileu−,leu−)derived from ATCC13059by long-term expose to ethyl-methane sulfonate(Best&Britz1986);ATCC13032 and its restriction-modification mutants,RM3and RM4(Schäfer et al.1994).The plasmid used was pCSL17,a7.2-kb E.coli-corynebacteria shuttle vec-tor(Hodgson et al.1989). C.glutamicum strains were grown routinely in Luria broth(Miller1972) supplemented with0.5%(w/v)glucose(LBG).Cells were cultivated at30◦C with agitation(200rpm)in a shaker incubator.Maximum specific growth rates (µmax)were determined from changes in A600for C.glutamicum strains in shake-flask cultures of LBG supplemented with up to8mg ml−1INH or10%(w/v) glycine and expressed as the percentage relative to growth in LBG.Preparation of cells and plasmid DNA for electroporationTwo media were used:LBG and LBG-GI(LBG con-taining2%glycine plus INH,which was used at 0.4mg ml−1for ATCC13032,RM3and RM4,or 4mg ml−1for AS019,MLB133and MLB194).Cells (100ml)were harvested at A600between0.35and 0.45,washed twice at4◦C with100ml of15% (w/v)of glycerol and then resuspended in2ml cold 15%(v/v)glycerol.pCSL17DNA were prepared by an alkaline lysis method(Britz&Best1986)and further purified with CsCl-EtBr density gradient cen-trifugation(Sambrook et al.1989).The source of heterologous DNA was pCSL17purified from E.coli strain LE392and homologous pCSL17was derived from C.glutamicum strain AS019.DNA was stored in aqueous solution at−20◦C.ElectroporationTransformation by electroporation was based on the protocol described by Haynes&Britz(1990)and this was carried out using a Gene-Pulser system(Bio-Rad Laboratories,Richimond,CA).Fortyµl of cell sus-pension(approximately109cells)were mixed with various amounts of DNA to give a total volume of 72µl and afinal concentration of15%glycerol.The mixture was held on ice for5min,then this was transferred to a pre-cooled0.2cm cuvette.Follow-ing exposure to a single pulse(2.5kV,25µF),the cells were immediately removed from the electrodes and stored on ice for5min.Subsequently,1ml of recovery medium(LBG medium containing10mM CaCl2and10mM mgCl2)was added to the cells.Fol-lowing incubation without shaking for1h at30◦C, 10-fold serial dilutions of cells were made in SMMC (Yoshihama et al.1985)and appropriate dilutions spread onto ET plates(Best&Britz1986)and ET containing50µg ml−1of kanamycin(ET-Km)to enu-merate transformants.Transformation efficiency(TE) was calculated as the number of transformants per109 cells electroporated perµg of plasmid DNA added and transformation frequency(TF)was calculated as the number of transformants per number of survivor per µg of DNA multiplied by106.Measurement of cell-wall thicknessCells were collected at an A600of0.4following growth in LBG,LBG-G(LBG with2%glycine),and LBG-INH(4mg ml−1).Thin sections offixed,em-bedded cells were viewed and recorded on a JEOL 100S electron microscope as described by Hayat (1981)and modified by Ms Anne Martsi Clintock (Dept.of Physiology,Monash University,Australia). Multiplefields were examined using several magni-fications and cells were photographed randomly.A magnification of12,000was used to measure cell-wall thickness,where10to20cells were viewed in each541parison of specific growth rates of C.glutamicum strains in the presence or absence of glycine or INH.Strains:AS019 ( ),MLB194( ),MLB133( )(left panels);ATCC13032( ), RM3( )and RM4( )(right panels).µmax values are expressed as a percentage of values seen for growth in LBG.frame.Cell wall thickness was measured for at least 40cells and multiple measurements(3–5)made for each cell,with averages plus standard deviations for each growth condition calculated using the Sigma-plot program.Determination of mycolic acid compositionStrains were grown to early exponential phase in shake-flask cultures of LBG and LBG-GI as described for electroporation experiments.The mycolic acid composition was determined by gas chromatography and mass spectrometry for cells and extracellularflu-ids following extraction,derivatization and quantifica-tion as described previously(Jang et al.1997).My-colic acids were quantified in terms of their peak area and the relative amounts expressed as a percentage of the total area detected for mycolic acids.ResultsSensitivity of strains to growth inhibition by glycine and INHStrains MLB133and MLB194were more sensitive to inhibition by both glycine and INH than AS019(rep-resentative of the parent of these strains),as indicated by their significantly lower relative specific growth rates when glycine was present at>1%and INH at >3mg ml−1(Figure1),confirming our previous ob-servations(Jang et al.1997).ATCC13032was more sensitive to both agents than AS019and both RM3 and RM4showed significantly more growth inhibition than their parent strain when grown in INH.In con-trast,the growth rates of ATCC13032,RM3and RM4 in glycine were similar,although RM3continued to grow in glycine at concentrations which completely inhibited the other two strains(>6%).Effect of glycine plus INH on mycolic acid profiles Our previous results had shown that the most signifi-cant impact of the presence of glycine plus INH during growth of the ATCC13059family of C.glutamicum strains was a significant increase in the proportion of extracellular mycolic acids(Jang et al.1997),where this effect was most noticeable for mutant MLB133 (Table1).The concentration of glycine used with the ATCC13032family of strains was kept at2%(w/v)in LBG but the concentration of INH had to be lowered to0.4mg ml−1to allow all strains to grow at accept-able rates.Even so,this level of glycine plus INH was very inhibitory,reflecting the greater sensitivity of these strains to the combination of these addi-tives.Although the profile of mycolic acids present in the ATCC13032family was similar to AS019and its mutants,the relative proportion of C32:0in all strains was lower than AS019and more resembled that seen for strain MLB133.Strain RM3was noted to contain relatively higher proportions of C36:2than ATCC13032and RM4,whatever the growth condi-tions used,which together with its very low levels of C32:0resulted in a higher ratio of unsaturated to saturated mycolic acids.Similar changes in mycolic acid composition were seen previously when strains MLB133and MLB194were grown in8mg of INH ml−1(Jang et al.1997),which suggests that strain RM3has undergone changes in its cell surface struc-tures which correlate with its greater sensitivity to INH.Growth in LBG-GI increased the proportion of extracellular mycolic acids in the ATCC13032family but the impact was not as great as seen for MLB133 and MLB194.However,there was no attempt to opti-mize the INH concentration used for the former and higher concentrations of INH may well have had a further impact.These results show that the concentra-tions of glycine and INH used altered the mycolic acid542Table1.Mycolic acid composition of C.glutamicum strains grown in two different media.Strains of AS019,MLB194and MLB133were cultured in LBG and were supplemented with2%glycine plus4mg INH ml−1and strains of ATCC13032,RM3and RM4in LBG were supplemented with2%glycine plus0.4mg INH ml−1.C32:053.0a55.942.139.046.150.840.843.528.930.547.466.6 C34:130.027.938.139.234.233.039.135.537.743.234.624.9 C34:08.810.67.18.68.5 6.27.29.38.0 2.68.5 4.5 C36:2 5.3 4.19.419.58.18.09.87.820.421.6 6.9 3.3 C36:1 2.934.4 3.313.8 3.2 2.1 3.0 3.9 5.0 2.2 3.2 1.0 %UM to TM b38.234.450.852.545.543.151.947.263.167.044.729.2 %Extracellular c 4.515.97.237.07.818.5 3.57.2 4.110.5 5.411.9543Fig.3.Transformation efficiencies seen following growth in LBG or LBG-GI for heterologous and homologous DNA.Strains were grown in either LBG or LBG containing2%(w/v)glycine plus INH(0.4mg INH ml−1for ATCC13032,RM3and RM4,or4mg INH ml−1for AS019,MLB133and MLB194)(LBG-GI)and trans-formed with pCSL17.Legend for numbers:1and3,LBG;2and4, LBG-GI.Transformation efficiency is expressed as the number of transformants per109cells electroporated perµg DNA supplied. TE(ranging from<10to103transformants×[109 cells×µg DNA]−1)for heterologously-derived DNA whereas homologous DNA gave much higher TE val-ues(1.7to3.1×104transformants×[109cells×µg DNA]−1).This indicates that these strains con-tain an active restriction system which impairs entry of foreign DNA.For all circumstances,the presence of glycine and INH in the medium improved both transformation ef-ficiency and frequency.For the ATCC13032family, the presence of2%glycine and0.4mg INH ml−1in the growth medium lead to TE increases of approxi-mately10-fold,indicating that transformation of the restriction mutants could be further improved by ma-nipulating the cell surface structure by prior growth conditions.Similar or greater increases in TE were also obtained for the AS019family,where growth in LBG-GI significantly improved transformation forheterologous DNA.Of the six strains tested,and the growth conditions used,the highest number oftransformants was obtained when MLB133cells weregrown in the LBG-GI(TE of5.8×106transformants ×[109cells×µg DNA]−1).The TE here was100-fold higher than for cells tranformed after growth inLBG for both sources of DNA and was between10-and100-fold higher than for AS019grown similarly.DiscussionAlthough we had previously reported the isolation and characterization of cell-surface mutants of C.glutam-icum(Best&Britz1986,Jang et al.1997),we had not reported their performance in plasmid transforma-tion.In this study,we confirmed that the cell surface mutants MLB133and MLB194were more sensitive than their parent to growth inhibition by glycine and INH,and that growth in glycine,INH or combinations of these caused an increase in the extracellular my-colic acids.Furthermore,analysis of cells of MLB133 by TEM showed that the cell surface was thinner fol-lowing growth in either glycine or INH.This was consistent with the observed increase in extracellu-lar mycolic acids and adds evidence to support our hypothesis that these agents are acting at the level of both synthesis and attachment of mycolic acids in C.glutamicum.Although MLB133still retained an active restric-tion-modification system,evidenced by its relatively poor transformation by heterologous DNA,this strain showed high electrotransformation efficiency relative to AS019,and this was comparable or better than the restriction and modification mutants,RM3and RM4,and their parent ATCC13032,even for heterol-ogous DNA.However,we noted that the ATCC13032 family of strains was more sensitive to growth inhibi-tion by glycine and INH,and we made no attempt to optimize the concentration of these to improve elec-trotransformation efficiency.These data point to the large variation between strains of C.glutamicum in sensitivity to growth inhibition by cell-surface modi-fying agents and the need to optimize conditions for each strain to improve transformation.This may arise due to differences in the cell surface structures present: we noted that the relative proportions of mycolic acids varied between the two families of strains studied here and suggest that these differences may have been544sufficient to explain the inherently better uptake of plasmid DNA by ATCC13032compared with AS019. It was also interesting and unexpected to note that strain RM3,which was isolated as a restriction minus mutant,showed a different mycolic acid profile to its parent strain ATCC13032in that it had a higher pro-portion of unsaturated mycolic acids.Changes in the surface structure may have added to this strains good performance in transformation.Also,the ATCC13032 family required less INH in the presence of glycine to have a significant impact on transformation efficiency, which was consistent with their greater sensitivity to these agents.These observations may help to ex-plain why other workers failed to see improvements in transformation of corynebacteria following growth in glycine or threonine(Dunican&Shivnan1989),as the concentration of glycine required to cause shifts in mycolic acid profile and sub-cellular location of these lipids varies considerably between strains and species of coryneforms.This study confirmed the presence of two barriers to successful introduction of DNA into C.glutamicum, where both the physical barrier of the mycolic acid-rich cell surface and the RM systems present play significant roles(Katsumata et al.1984,Haynes& Britz1990,Schäfer et al.1994).It also provided an opportunity to better understand the relative con-tribution of these two barriers by showing that the uptake of DNA by restriction-minus mutants could be further improved by manipulating their cell surface structures.Our analysis concentrated on the impact on glycine and INH on mycolic acid structures.How-ever,it is interesting to speculate that the observed cell surface changes in mutant MLB133may also have affected restriction and modification activity in this strain,given the observed large increase in trans-formation efficiency for heterologous DNA following growth in LBG-GI.Schäfer et al.(1994)described a stress-sensitive restriction system in C.glutam-icum which was impaired by heat shock or treatment with surface active agents(detergents and ethanol), where the latter implied that the enzymes concerned may have been located at or near the surface of the cells.Shedding of mycolic acids into the extracellular growth medium during growth in glycine plus INH may alter the activity of this system by mechanisms which may include changes in the lipid environment necessary for activity or concurrent release of the en-zyme(s)concerned into the extracellular medium.This remains experimentally uninvestigated but warrants further study.AcknowledgementsThe authors gratefully acknowledge Drs Schäfer,Kali-nowski and Prof Pühler for generously providing strains ATCC13032,RM3and RM4.ReferencesBest GR,Britz ML(1986)Facilitated protoplasting in certain aux-otrophic mutants of Corynebacterium glutamicum.Appl.Micro-biol.Biotech.23:288–293.Billman-Jacobe H,Wang L,Kortt A,Stewart D,Radford A (1995)Expression and secretion of heterologous protease by Corynebacterium glutamicum.Appl.Environ.Microbiol.61: 1610–1613.Britz ML,Best GR(1986)Expression of chloramphenicol resis-tance by plasmid pHY416hosted in Corynebacterium glutam-icum.Curr.Microbiol.14:13–17.Dunican LK,Shivnan E(1989)High frequency transformation of whole cells of amino acid producing coryneform bacteria using high voltage electroporation.Bio/Technology7:1067–1070. Hammes K,Schleifer KH,Kandler O(1973)J.Bacteriol.116: 1029–1053.Hayat MA(1981)Mode of action of glycine on the biosyn-thesis of peptidoglycan.Principles and Techniques of Elec-tron Microscopy-Biology Applications,2nd A:Edward Arnold.Haynes JA,Britz ML(1990)The effect of growth conditions of Corynebacterium glutamicum on the transformation frequency obtained by electroporation.J.Gen.Microbiol.136:255–263. Hodgson ALM,Haynes JA,Britz ML,Nisbet IT(1989)Construc-tion and utilisation of a Corynebacterium-Escherichia coli shut-tle vector.In:Proceedings,VIIIth Biotechnology Conference, Sydney,Australia,pp.181–184.Hopwood DA Wright HM,Bibb MJ,Cohen SN(1977)Genetic re-combination through protoplast fusion in Streptomyces.Nature 268:171–174.Jang KH,Chambers PL,Britz ML(1996)Analysis of nucleotide methylation in DNA from Corynebacterium glutamicum and related species.FEMS Microbiol.Lett.136:309–315.Jang KH,Pierotti D,Kemp GW,Best GR,Britz ML(1997)Mycolic acid composition of Corynebacterium glutamicum and its cell surface mutants:effects of growth with glycine and isonicotinic acid hydrazide.Microbiology143:3209–3221.Katsumata R,Ozaki A,Furuya A(1984)Protoplast transformation of glutamate-producing bacteria with plasmid DNA.J.Bacteriol.159:306–311.Liebl W,Bayerl A,Schein B,Stillner U,Schleifer KH(1989) High efficiency electroporation of intact Corynebacterium glu-tamicum.FEMS Microbiol.Lett.65:299–304.Miller JH(1972)Experiments in Molecular Genetics.Cold Spring Harbor,NY:Cold Spring Harbor Laboratory Press. Sambrook J,Fritsch EF,Maniatis T(1989)Molecular Cloning:A Laboratory Manual,2nd edn.Cold Spring Harbor,NY:Cold Spring Harbor Laboratory Press.Schäfer A,Schwarzer A,Kalinowski J,Pühler A(1994)Cloning and characterization of a DNA region encoding a stress-sensitive re-striction system from Corynebacterium glutamicum ATCC13032 and analysis of its role in intergeneric conjugation with Es-cherichia coli.J.Bacteriol.176:7309–7319.545Smith MD,Flickinger JL Lineberger DW,Schmidt B(1986)Proto-plast transformation in coryneform bacteria and introduction of α-amylase gene from Bacillus amyloliquifacients into Brevibac-terium lactofermentum.Appl.Environ.Microbiol.51:634–639. Tomiyasu I,Yano I(1984)Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa.Arch.Microbiol.137:316–323.Walker RW,Prome JC,Lacave CS(1973)Biosynthesis of mycolic acids formation of a C32β-keto ester from palmitic acidsin a cell-free system of Corynebacterium diphtheriae.Biochim.Biophys.Acta326:52–62.Yoshihama M,Higashiro K,Rao EA,Akedo M,Shanabruch WG, Follettie MT,Walker GC,Sinskey AJ(1985)Cloning vec-tor system for Corynebacterium glutamicum.J.Bacteriol.162: 591–597.。