Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

AGRICULTURE, AGRIBUSINESS AND BIOTECHNOLOGY

Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

Sangiliyandi GurunathanI, ^*; Paramasamy Gunasekaran^II

^IDepartment of Cell and Developmental Biology; School of Medicine; University of North Carolina at Chapel Hill; Chapel Hill NC 27599-7090; 108, Issac M Taylor Hall; North Carolina - USA

^IIDepartment of Microbial Technology; School of Biological Sciences; Madurai Kamaraj University; Madurai 625 021; India

ABSTRACT

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB.

Key words:Zymomonas mobilis, Escherichia coli, Expression, Levansucrase, Extracellular sucrase

RESUMO

Os genes do sacB e do sacC que codificam o levansucrase e o sucrase extracellular respectivamente foram sub-clonados independentemente no pBluescript (número elevado de cópias) e no vetor ponte para Z. mobilis-E. coli, pZA22 (baixo número de cópias). A expressão destes genes foi comparada tanto em E. coli quanto em Z. mobilis. O nível da expressão do gene sacB em E. coli foi quase dez vezes menor do que a expressão do gene sacC, independente do meio de cultivo ou da linhagem hospedeira. Em Z. mobilis, a expressão dos genes sacB e sacC mostrou regulação dependente da fonte de carbono. Os níveis dos transcritos sacB e sacC estavam três vezes mais aumentados em células cultivadas em sacarose do que aquelas cultivadas em glicose/frutose. A análise por Northern blot revelou que o nível do transcrito sacC era 2-3 vezes maior do que aquele de sacB. Esses resultados sugerem que a expressão do gene sacC foi mais pronunciada do que sacB.

INTRODUCTION

Zymomonas mobilis, a gram negative bacterium, has been shown to produce ethanol at a rate three to four fold, and at a higher final yield compared to the traditionally used yeast strains (Rogers et al., 1982). However, this organism uses only a narrow range of substrates, which is limited to glucose, fructose and sucrose (Swings and DeLey, 1977). However, when grown on media containing sucrose as the carbon source, the ethanol yield is considerably reduced because of the by products formed such as levan, fructo-oligomers and sorbitol (Viikari, 1984). When Z. mobilis is grown on sucrose medium, extracellular sucrose hydrolyzing enzymes such as intracellular sucrase (SacA or InvA), extracellular levansucrase (SacB or Inv C) and extracellular sucrase (SacC or InvB), are produced . The SacA enzyme is a monomer with a molecular weight of 58 kDa. The sacA gene from Z. mobilis has been cloned, sequenced and characterized (Gunasekaran et al., 1990a). The deduced amino acid sequence of sacA gene product showed strong homology with the intracellular sucrase of Bacillus subtilis and yeast invertases. The intracellular sucrase SacA from Z. mobilis was purified to homogeneity from a recombinant E. coli strain containing the sacA gene under an expression system. The protein was monomer with a molecular mass of 58kDa. The sucrase activity was maximal at 25º C and thermal stability of the purified protein was low and maximal activity was at pH 6.5 Therefore, SacA alone is unable to support the growth of Z. mobilis on sucrose (Ait-Abdelkader et al., 2000).

Levansucrase, a beta-fructo-furanosidase, is an extracellular enzyme that is responsible for both sucrose hydrolysis and levan formation. Levansucrase has been purified from Z. mobilis cells (Lyness and Doelle, 1983) and from culture broth (Mortatte et al., 1983). It is a monomer with a molecular weight of 51 kDa in Z. mobilis ZM4 (Preziosi et al., 1990b). The SacB enzyme purified from culture filtrate of Z. mobilis Z6C has a molecular weight of 56 kDa (Yanase et al., 1992). The gene encoding (sacB) levansucrase and (sacC) in Z. mobilis NRRL B.806 and ATCC10988 has been cloned and sequenced (Gunasekaran et al., 1995). Recently, the over expressed levansucrase (SacB) was purified from the recombinant E. coli by a simple method and characterized (Sangiliyandi and Gunasekaran, 1998). A levansucrase (SacB) was purified to elecrophoretic homogeneity from a recombinant E. coli. The 55 kDa enzyme hydrolyzed b-fructosides but not a glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity was at 30º C and hydrolase activity at 50º C. However, the polymerase activity was selectively abolished by para-chloromercuribenzoate (Sangiliyandi et al., 1999a) and also by in-vitro mutagenesis (Sangiliyandi et al.,1999b). Recently, a levansucrase mutant was isolated by in-vitro mutagenesis using hydroxylamine. By mutation in the protien, the levanforming activity was abolished but the sucrase activity was retained in the mutant strain. This uncoupling of the sucrase activity from the levan-forming activity suggests a change in the protein conformation due to mutation and elevated temperature could have led to the loss of activity (Sangiliyandi and Gunasekaran, 2001).

An extracellular sucrase (Sac C or InvB) has a high specific activity for sucrose hydrolysis. Nearly this enzyme contributes 60% of the extracellular sucrase activity. However, it catalyses neither fructose polymerization into levan nor degradation of polyfructose such as levan or inulin. Thus this enzyme differs from the B. subtilis SacC, which shows levanase activity in addition to sucrose hydrolysis (Kannan et al., 1995). The purified active enzyme from Z. mobilis strain CP4 is a monomer with a molecular weight of 46-47 kDa (O Mullan et al., 1992). The sacC gene was cloned and expressed in E. coli (Kannan et al., 1995). The overexpressed extracellular sucrase of Z. mobilis was purified from a recombinant E. coli by repeated cycles of freezing and thawing, followed by hydroxyapatite column chromatography. The purified enzyme had a Mw of 46kDa, as determined by SDS-PAGE. Its km value for sucrose was 86mM and had an optimal pH and temperature at 5.0 and 36^oC respectively (Sangiliyandi and Gunasekaran, 2000). Here we show the differential expression of levansucrase and extracellular sucrase activity of Z. mobilis sucrase genes in E. coli and Z. mobilis hosts.

MATERIALS AND METHODS

Bacterial strains and plasmids

Escherichia coli^,JM109 (RecA1, endA1, gyrA96 thi, hsdR17, SupE, relA1(lac^-proAB) [F' traD36 proAB, lacIq, D M15] BL21(DE3) F^-hsdS gal (r^-m^-) lon^-ompT. XL1 Blue EndA1 hsd17 (r-m⁺) supE44 thi-1 l -gyrA96 recA1 D lac (F' proAB lacIq D M15 Tn10). Plasmid pBluescript KS⁺(Stratagene, USA). Z. mobilis wild type (NRRL B-806). The recombinant plasmid pLSS41 carrying 5 kb chromosomal DNA fragment coding for sacB and sacC of Z. mobilis. Plasmid pRK2013 was used as a helper for conjugations.

Growth conditions

Z. mobilis strains were grown in complete media RM containing (per liter) glucose 20 g, yeast extract 10 g, KH₂PO₄ 2 g and pH adjusted to 6.0. Whenever necessary, different carbohydrate sources such as glucose (RMG) or fructose (RMF) or sucrose (RMS) was added to RM. The cultures were grown at 30ºC at static condition. E. coli strains were grown at 37ºC under agitation in LB or M63 medium.

Construction and screening of recombinant sucrase clones

A 1.7 kb DraI fragment containing an ORF encoding levansucrase (SacB) from the plasmid pLSS41 was purified by the method of Suzuki et al. (1991) and then ligated with EcoRV digested pBluescript KS⁺. The resultant plasmid pLS3 had only sacB gene located downstream of the T7 promoter in the pBluescript KS⁺. This recombinant plasmid was transformed into E. coli JM109. A 2.1 kb Pvu II fragment containing an ORF encoding levansucrase (SacB) from the plasmid pLS3 carrying sacB was purified and cloned into Eco RV site in the shuttle vector pZA22. The resultant plasmid was designated as pLSP13. A 2.1 kb Pvu II fragment containing an ORF encoding extracellular sucrase (SacC) from the plasmid pLSS41 carrying sacC was purified and cloned into the Eco RV site of pBluescript KS⁺. The resultant plasmid was termed pZS63. A 2.1 kb Pvu II fragment containing an ORF encoding extracellular sucrase (SacC) from the plasmid pLS63 carrying sacC was purified and cloned into the Eco RV site of the shuttle vector pZA22. The resultant plasmid was termed pZSP62.

Assay of levansucrase activity

Sucrose hydrolyzing and levan forming activities were assayed as previously described (O' Mullan et al., 1992). Levan was estimated by hydrolyzing the levan in 0.1 N HCl at 100 ^oC for 1 h according to Viikari and Gisler, (1986) and expressed as fructose units. One unit of levan forming activity of the levansucrase was defined as the amount of enzyme required to produce 1 µmole of levan (as fructose units) in one minute. One unit of sucrose hydrolysing activity of the levansucrase was defined as the amount of enzyme releasing 1 m mole of reducing sugar per minute. The protein concentration was determined by the method of Lowry et al. (1951).

Recombinant DNA techniques

Preparation of plasmid DNA was carried out by standard method (Sambrook et al.,1989). Purified plasmid was obtained using plasmid purification kit (Qiagen Inc., USA). The DNA fragments were separated as mentioned by Suzuki et al. (1991). All cloning experiments were carried out according to standard methods (Sambrook et al., 1989). Transformation was performed as described by Chung and Miller, (1988).

Bacterial conjugations

Bacterial conjugations were performed by triparental filter matings as previously described (Arvanitis et al., 2000). For counter selection against donor cells, 100mg/ml nalidixic acid was used for ATCC 10988. All transconjugants were tested for their plasmid content with plasmid isolation, back transformation, and restriction analysis.

Dot Blot hybridization

Northern Dot Blots containing upto 5ug of alkaline —denatured total RNA were prepared using 9x12cm Zeta Probe GT Nylon membranes and a Bio Dot SF Microfiltration apparatus (Bio-Rad 170-6543). Membranes were hybridized using Express Hyb Hybridization solution (Clontech), and DNA probes end radiolabeled by using (g 32 P) dATP (3000Ci/mmol) or by direct incorporation using (a -32P) dCTP (3000Ci/mmole) Amersham redivue). Membranes were washed twice for 30min at 61ºC in 1mM EDTA , 40mM NaHPO_4, pH 7.2 and 5% according manufacturers instructions. RNA signal in the same sample using phosphorimager analysis and the data acquisition packages provided with instrument.

Northern Blot hybridization

Total RNA of Z. mobilis and E. coli was isolated as described previously (Ausubel et al., 1994). Samples (5µg) were electrophoresed on a 0.8% agarose-formaldehyde gel and transferred to a nylon membrane filter (Schleicher and Schuell). Pre-hybridization and hybridization was carried out in hybridization solution (50% formaldehyde, 5x Denhardt reagent, 50mM sodium phosphate (pH5.5), 120 mM NaCl, 0.1% SDS, 125g of sonicated salmon sperm DNA per ml) at 50º C. The gene specific probes were labeled with random-primed labeling kit (Boehringer Mannheim). Filters were washed twice at room temperature and twice at 65º C in 2X SSC-0.5% SDS and then washed once at room temperature in 0.1x SSC. These filters were exposed to X-ray films.

RESULTS AND DISCUSSION

Subcloning of sucrase genes of Z. mobilis in E. coli

In order to study the promoter strength of levansucrase and extracellular sucrase genes, the sacB gene and sacC gene were cloned and expressed in E. coli. Z. mobilis chromosomal DNA fragment (a 5.0 kb HindIII fragment) present in the plasmid pLSS41 possess ORF1 and ORF2 that encoded extracellular levansucrase (SacB) and extracellular sucrase (SacC) respectively (Gunasekaran et al., 1995). The levansucrase gene sacB resides within the two DraI restriction sites separated by 1.7 kb. The purified 1.7 kb fragment was cloned into EcoRV site of pBluescript KS⁺. The ligated mixture was used to transform E. coli JM109. The transformants were selected on LB agar containing ampicillin (20 mg/l), IPTG (40 mg/l) and X-gal (20 mg/l) by Blue-white selection. All the white colonies were scored as recombinant clones. The recombinant clones were independently grown in M63 medium supplemented with glycerol (1% v/v), cell lysate was prepared and assayed for both sucrose hydrolyzing activity and levan forming activity. The cellular fractions of all recombinant clones exhibited sucrase activity and levan forming activity on standard plate screening. Plasmid was isolated from a recombinant clone and was designated as pLS3. The plasmid pLS3 was used to transform E. coli JM109. All the transformants exhibited levansucrase positive phenotype i.e. sucrose hydrolyzing and levan forming activities, confirming the plasmid pLS3 carried DraI fragment encoding levansucrase. The same selection procedure was followed for screening of pZS63 plasmids.

2.1 kb Pvu II fragment in pLS3 carrying sacB was isolated and cloned into EcoRV digested pZA22. The ligated mixture was used to transform E. coli JM109. Transformants were selected on LB agar containing Cm (30 µg/ml) and replica plated on LB agar containing Tc (15µg/ml). The Cm^r Tc^s colonies were selected as recombinant clones and were analyzed for levansucrase activity. Plasmid from one of the levansucrase positive clone was isolated and designated as pLSP13. For subcloning of sacC gene, a 2.1 kb PvuII fragment from pZS63 was isolated and ligated with EcoRV linearized pZA22. The ligation mixture was used to transform E. coli JM109. Transformants were selected on LB Cm (30 µg/ml) plates, replica plated on LB Tc plates and tested for sucrose hydrolysis activity as described earlier. One of the sucrase positive clones carried a recombinant plasmid, designated as pZSP62. The presence of the 2.1 kb insert in this plasmid was confirmed by restriction analysis. This plasmid was 8.8 kb in size and carried sacC gene downstream to T7 promoter.

Expression of Sucrase genes in E. coli

The plasmids pLSP13 and pZSP62 (in pZA22, shuttle vector) were separately transferred into the E. coli strains JM109 and XL1Blue in which the sacB and sacC genes use their own promoter for expression. Comparison of the recombinant strains grown in LB medium revealed that the E. coli strain carrying sacB gene exhibited sucrase activity of 0.24-0.27 U/mg while the strains carrying sacC gene exhibited sucrase activity of 3.03-3.10 U/mg. Similar comparison of the strains grown in M63 medium showed that the sucrase activity of the strain carrying cloned sacB gene was 0.60-0.63 U/mg while the strain carrying cloned sacC gene expressed an activity of 7.04-7.10 U/mg. These results suggested that, the level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain (Fig .1.)

The plasmids pLS3 and pZS63 (in pBSKS⁺) were independently transferred into E. coli strains and the expression of sacB and sacC genes were studied under the conditions of with and without IPTG. The cloned genes were expressed using T7 as well as the native promoter in E. coli. Analysis of the LB grown cells revealed that E. coli (pLS3) produced a low level of sucrase activity (0.56 —0.60 U/mg) than the E. coli (pZS63) (5.79-5.80 U/mg). Comparison of the cells grown in different media revealed that the M63 medium grown cells produced higher level of sucrase activity. In this medium, the sacB gene carrying cells produced sucrase activity of 1.12-1.20 U/mg while the sacC gene carrying strains exhibited sucrase activity of 9.15 —9.20 U/mg. These results suggested that the expression of sacB was almost 10 fold lower than that of the sacC gene, when it was expressed by its own promoter.

Comparison of induced expression of sacB and sacC genes in E. coli BL21

As we expected, the previous experiments showed that the expression of SacC was more than SacB by its own promoter. Then we examined the E. coli (carrying plasmids for sacB and sacC in which the genes were cloned under T7 promoter) cultures were grown in M63 medium with glycerol or fructose till they reached 1 OD at A₆₀₀. IPTG (400 µM) was added to the cells and harvested after 4 h of incubation at 37ºC. Addition of IPTG to the growing cultures of recombinant E. coli BL21 induced the expression of sacB or sacC genes from all plasmids (pLS3, pLSP13, pZS63 and pZSP62). The uninduced culture of E. coli (pLS3) had sucrase activity of 4.5 U/mg, which after induced expression of sacB gene resulted in sucrase activity of 72.0 U/mg (Table.1). The uninduced cells carrying similar plasmid construct with sacC gene (pZS63) had a high level of sucrase activity (25.6 U/mg) which upon induction produced a very high level (>100 fold) of sucrase activity (2672 U/mg). Similar experiment was carried out with the strains carrying pLSP13 and pZSP62. The uninduced cells carrying sacB gene had a low level of sucrase activity (1.9 U/mg) but after induction showed a ten-fold increase in sucrase activity (19.0 U/mg). In contrast, the uninduced cells carrying sacC gene had sucrase activity of 12.8 U/mg, which after induction resulted in more than 100-fold increase in sucrase activity. These results supported that the sacC gene responded better than the sacB gene, in the presence or absence of IPTG expression.

Isolation of sucrase negative mutant of Z. mobilis

A highly stable extracellular levansucrase, extracellular sucrase and intracellular sucrase mutant (SNM10) of Z. mobilis strain B-806 was isolated after NTG mutagenesis (50µg/ml for 45 min at 30º C) from the strain LS1A (Kannan et al., 1998). It was repeatedly sub cultured in glucose medium and plated on RM agar plates. The cellular extracts of the randomly selected colonies were subjected to zymogram analysis for the detection of sucrase activity. Some of the isolate did not produce the sucrase band corresponding to SacA, SacB and SacC and it was designated as SNM 1-10. Among 10 isolates, one of the isolate was taken for further studies. For confirming the stability of the SacA, SacB and SacC mutation, the strain (SNM10) was subcultured (3 times, 18 h growth period) in RM glucose medium, and then plated on RM agar plates. Randomly selected colonies were grown in RM glucose/fructose medium and the cellular fractions were analysed on native -PAGE and by zymogram staining (data not shown). None of the culture showed any reversion of the SacA, SacB and SacC mutation. As this strain was derived from the strain LS1A, the cellular fraction did not exhibit sucrase band corresponding to levansucrase (SacB) and intracellular sucrase (SacA). Therefore, the mutant LS1A was phenotypically defective for the synthesis of levansucrase and intracellular sucrase. The novel complete sucrase negative mutant SNM10 was phenotypically defective for the synthesis of all sucrases. We used this mutant (SNM10) for the expression of each plasmid encoded sucrase genes in Z. mobilis.

Expression of sucrsae genes in E. coli and Z. mobilis

We examined the effect of plasmid encoded sucrase synthesis in E. coli and different sucrase mutants of Z. mobilis. The plasmid were transformed in to Z.mobilis as described in Material asnd Methods section. Table 2 shows the extracellular sucrase activities exhibited by extract of E. coli and Z. mobilis. Strain B-806 was expressing sucrase genes by chromosomal encoded but the mutants strains of Z. mobilis were expressing by plasmid encoded sucrase genes. Most of the extracellular sucrase activity (nearly 70%) produced by sacC gene and remaining activity was produced by sacB gene in Z.mobilis, as well as the same result was also observed in E. coli strains. Previous experiments have shown that the sacC gene carrying E. coli strain caused dramatic increase in the functional expression. The same effect was observed in E. coli and recombinant Z. mobilis strains.

We successfully introduced sucrase genes for synthesis of sucrase in wild type and sucrase negative mutants by expressing the Z. mobilis genes encoding sacB and sacC separately. Under the control of the each gene promoter, the specific activities of these two enzymes ranges from 2.0 to 4.3 U/mg (Table 2). However, both the genes were expressed successfully in all strains. The level of expression of the enzymes by plasmids was considerably higher than the levels of expression observed in chromosomal encoded. The difference between the levels of expression may be a function of a plamid copy number and of transcript coupled with and protein size and stability.

Transcriptional analysis of sacB and sacC genes

According to the previous experiments, the sacC gene was expressed more stronger than sacB gene, irrespective of the medium and strains. we wanted to examine the same effect of sacB and sacC gene in E. coli as well as Z. mobilis by transcriptinal analysis. Hybridization of RNAs from Z. mobilis and E. coli (carrying sacB and sacC genes) cells, which were grown on glucose, fructose and sucrose was performed in order to determine whether enzyme induction was the result of transcriptional regulation of sucrase genes by carbon sources or the effect of promoter. The bands of 1.5 and 2.9 kb in size were also detected from cells grown on media containing glucose or fructose and sucrose and also E. coli cells grown minimal media. From these results, we concluded that sacB and sacC genes were expressed constitutively at the basal level (data not shown).

Dot blot analysis

In order to determine, if the high level expression of SacC was due to its strong promoter or was influenced by any carbon sources. Dot blot analysis of sucrases of mRNA levels in Z. mobilis cells grown on glucose, fructose and sucrose was performed. Surprisingly, the levels of sucrase mRNA of Z. mobilis cells grown on sucrose were found to be approximately three fold higher than the levels observed in cells grown on glucose and two fold higher than the levels observed in cells grown on fructose. Similar cases have been postulated in the gene expression of Z. mobilis and also depends on carbon source dependent regulation (Liu et al., 1992) It was of interest to determine if the increased level of transcript in sucrose grown cultures was also a general phenomenon for the expression of sucrase genes. Filters prepared with RNA samples were subjected to hybridization with sacB and sacC gene specific probes . Strong hybridization signals were detected for the RNA samples hybridized with sacC probes than sacB probes. Among the RNA samples from the cultures grown in different carbon sources, the maximum sucrase transcript was from the culture grown in sucrose (Figs. 2a and 2b). These experiments showed that the high amount of specific mRNA were present in sucrose grown culture than glucose and fructose grown cells.

NORTHERN ANALYSIS

To compare the expression of plasmid encoded sacB and sacC genes in Z. mobilis, and recombinant E. coli strains, the RNA samples from all cultures were subjected to northern hybridization analysis using specific probes for sacB and sacC. While hybridized with sacB gene probe, strong signals were detected for the RNA samples of Z. mobilis and E. coli (pLS3) but relatively faint signal was observed for RNA samples from E. coli (pLSP13). Quantitative analysis of multiple hybridization experiments consistently showed two fold higher level of transcripts detected in Z. mobilis and E. coli (pLS3) than in E. coli (pLSP13). The sacB probe did not hybridize with sacC transcript from E. coli (pZS63) (Fig. 3a.). Similarly the sacC specific probe did not hybridize with the transcript from levansucrase clones i.e. E. coli (pLS3). The hybridization of sacC probe with the RNA isolated from Z. mobilis and E. coli was studied. The levels of sacC mRNA levels in E.coli (pZS63) was three fold higher than E.coli (pZSP62) and two folds higher than Z. mobilis (Fig. 3b). The SacC probe did not hybridize with transcript from E. coli (pLS3).

Comparison of the sacB and sacC transcripts, in the above strains revealed that the level of sacC transcript was approximately three fold higher than sacB transcript in Z. mobilis and also E. coli expressed sacB gene. Comparison of the transcript levels in the strains [ E. coli (pLS3) with (pZS63) and E. coli (pLSP13) with E. coli (pZSP62)] revealed that the sacB transcripts were much lower than the sacC transcript. These results suggested that the expression of sacB in either Z. mobilis or E. coli was much lower than the expression of sacC gene, probably due to inefficiency of sacB promoter. However, it is observed that extracellular sucrase (SacC) was unstable than extracellular levansucrase (SacB) (Sangiliyandi and Gunasekaran, 1998; Sangiliyandi et al., 1999a; Sangiliyandi et al., 1999b).

A high relative abundance of sacC specific transcripts might compensate for the instability of the Z. mobilis extracellular sucrase. Similar result was observed with the polycistronic mRNA of the Z. mobilis glf, zwf, edd, glk operon. Liu et al (1992) suggested that a high relative abundance of glk specific transcripts might compensate for the instability of the glucokinase. Eddy et al. (1989) reported that the ratio of gap message to pgk message was approximately 5:1 ratio. The differential expression of gap and pgk appears to be controlled at the mRNA level.

The pattern of codon usage in sacB gene was compared with two classes of genes known in Z. mobilis - highly expressed genes like pyruvate decarboxylase (pdc), glucose-6-phosphate dehydrogenase (zwf) and poorly expressed genes like intracellular sucrase (SacA), acid phosphatase (PhoC) (Pond et al., 1989) and ligase (lig) (Gunasekaran et al., 1990a). The codon bias was calculated according to Shark and Conway (1992). It was found that the codons in sacB strongly correlated with codon usage and bias with poorly expressed genes. The three codons TGC, CTA and TAC were not used in sacB. The codon TGT only was used for cys in sacB. In sacB the preference of codons AAT, GAT, CAT and AAA were similar to low expressed genes. However, the use of codon CAG and TAT in sacB was similar to highly expressed genes. Thus, sacB could be classified as one of the low to intermediately expressed genes of Z. mobilis.

The relative abundance of the SacC enzyme in Z. mobilis might be due to high levels of sacC mRNA, a high rate of translation of the sacC message, or resistance of the sacC to proteolysis. Since most of the enzymes remain at high level at stationary phase, this factor alone can not be accounted for the high SacC activity. Therefore, the high level of extracellular sucrase results from an abundant sacC message, perhaps due to a combination of rapid transcription and significant mRNA stability. Stability of the sacC message might be imparted by the substantial stem-loop structures located both upstream and downstream of the sacC gene as suggested for several other Z. mobilis genes. In Northern blot analysis, increased expression of the SacC in cells grown on sucrose was proven to be the result of an increased transcription rate, compared with cells grown on glucose and fructose (Figs. 3a and 3b). Similar results were also observed in the gene expressions of Z. mobilis; carbon source dependent regulation (Liu et al., 1992). Considering the amount of transcripts, both the genes in the operon seemed to be expressed differently from each other, because of the existence of terminator sequence of sacB and sacC gene (Song et al., 1999). Similarly, Newbury et al. (1987) postulated that an intercistronic transcription terminator could act as a gene expression regulator in the analysis of a polycistronic malEFG operon of E. coli. Northern blot analysis suggested that the levels of sacC transcript were approximately three folds higher than sacB transcript in Z. mobilis as well as the E. coli (pZS63 or pZSP62). Thus, it is surprising that, the gene dosage can improve the hydrolysis of sucrose substrates during bioconversion to ethanol. Optimization of such a system by manipulating the growth conditions and genetically modifiying the host metabolism as well as development of ethanol retrieval technologies for the growth medium, could lead to production of ethanol by this organisms at an industrial level.

ACKNOWLEDGEMENTS

This work was supported by grants (No. 38/0912/EMR II) from CSIR, New Delhi and (No. SP/SO/A52/96) from DST, New Delhi. GS thanks UGC, New Delhi for their support with Senior Research Fellowship. We thank Mr. Arul Subramanian for critically reading this manuscript.

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Received: September 11, 2002

Revised: July 28, 2003

Accepted: February 27, 2004

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*

Author for correspondence

Publication Dates

• Publication in this collection
  03 Aug 2004
• Date of issue
  July 2004

History

• Accepted
  27 Feb 2004
• Received
  11 Sept 2002