- Email: [email protected]
Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum
MICROBIOLOGY LE-I-I-ERS
ELSEVIER
FEMS Microbiology
Letters I37 ( 1996)
I IS I2 I
Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum *, Victor
Annette M. Griffin Received 23 November
J. Morris, Michael J. Gasson
1995; revised 22 January
1996; accepted 23 January
1996
Abstract The aceA gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of gumD from Xanthomonas campestris. Therefore aceA is likely to encode the phosphate-prenyl glucose l-phosphate transferase catalyzing the first step in acetan biosynthesis in A. xylinum. Kqwwdc:
Gene: Acetan; Cloning:
DNA sequencing;
Exopolysaccharide;
1. Introduction Many microorganisms have the ability to synthesize extracellular polysaccharides (EPS) and excrete them from the cell, as either soluble or insoluble polymers. Such polymers confer selective advantages on the host; for example, allowing adherence to inert surfaces, protection from desiccation and other environmental stresses, antigenic variation of pathogenic strains, and masking immune recognition. Consequently bacterial EPS provide important model systems for the study of molecular assembly and secretion, gene regulation, cell-cell interactions, symbiosis, pathogenesis, and the relationship between polysaccharide structure and function [1,2]. In addition, microbial polymers, such as xanthan, are widely used in the food and other industries as gelling,
* Corresponding author. Tel.: +44 (1603) 255 000 or 255 354; Fax: + 44 (I 603) 507 723; E-mail: [email protected] 037% 1097/96/$15.00 0 1996 Federation PII SO378- 1097(96)00049-3
of European
Microbiological
Acetohacter
xylinum
thickening, texturizing, suspending and encapsulating agents [3]. In recent years there has been considerable interest in developing novel microbial polysaccharides for use in industry and one of the long-term aims of these studies is to genetically engineer new polymers with altered structures that confer improved and novel properties. Acetobacter xylinum is used in the production of a sweet confectionery (Natal enjoyed in many areas around the world [4]. This organism produces two different extracellular polysaccharides: cellulose and acetan. Acetan is an acidic polysaccharide which is structurally related to xanthan [5]. In order to engineer novel acetan based polysaccharides it is essential to genetically characterize the pathway for biosynthesis. Although the biochemical pathway for acetan biosynthesis has been elucidated [6], nothing is known of the genetics of this process. This paper describes our strategy for the identification, cloning and sequencing of the A. xylinum aceA gene predicted to encode the first step in acetan biosynthesis. Societies. All rights reserved
2. Materials
and methods
2. I. Media and reagents The Cl strain of A. xylinum was grown in AJ medium at 30°C as described previously 171. Stains of Escherichia coli strains were grown in L-broth or on L-agar [8], or NZYCM media [9] at 37°C. Ampicillin was added to a concentration of 100 pg ml-’ when required. Strains for plating lambda phage were grown in L-broth containing 0.2% maltose and 10 mM MgSO, at 30°C as described [9]. Lambda were stored in SM buffer [9] at 4°C.
@age 1
2.2. Bacterial strains and plusmids A library of A. xylinum strain Cl was constructed in lambda DASH II (Stratagene, Cambridge, UK). E. coli XLI-Blue P2 (Stratagene) was used as the host for phage lambda and strain TGl [9] was used as a host for plasmids. 2.3. DNA manipulations Restriction enzymes and DNA modifying enzymes were purchased from Gibco BRL, Paisley, UK and were used according to the manufacturer’s
35 -TTTCATGAAT
TGTCCGTGGTTTTGTCTTTTCTGGCCTCATATCGTTACGA
'ATTGGGAAATTC
100
101
CTTCCATGTATTGATGTCGTTATTTTTCGCCTCCAAA
200
201
AAATGGGGATTATTTCGCACTCGATn;TGCAGTGCGGA MGIISHSIVQCGKWQAGPARRQVVFRVHRRCFK
300
301
AATGAGCGAGGCACTCAGGCACTTGAAGAATGGCGAAACG~CGCTACCAT~CGGTCTCGA~GCATTCCC~~GCATCC~TAC~GCG~ MSEALRHLKNGETDLSSLPSGLDSIPRRIHTAR
400
401
GGCGTGGATGACAGCTTTTCCTACCGGCATCCTGTCCTGC GVDDSFSYRHPVLPQVVVLSDAFCVCLAVVACIA
500
501
CCTGGCAGAGGATATACGATACCCCCGATGAACTGTCTGGT WQRIYDTPDELSGALLLANIMAAGAFFLFPKNV
600
601
TCCCCTCCTCGACATTCCCGCATAACCAAGGTTTCTGTC PLLDIPDITKVSVQIRYLLPPVAFGEVVFCTVL
700
701
GTCATGCTGTCATGGCCGTTTGGCGTGACGGTGCGCATGG VMLSWPFGVTVRMGMEWLTFVVAILFVERCVGTY
800
801 901
GCGCATGCAGCGCATGTTCCGCCTCGTCGGGACATTTGAT RMQRMFRLVGT FDDQGGDSDGTVEELVLRARED CATATCGATGCGGTCATCATATGCTTTCCCCCCGCGTGCG HIDAVIICFPPACGQQHVMDVLWRLRGVLADVYV
1100
1301
GACTCCCGCGGGCCGGTGCTGTTCTGCCAGCCCCGGCAGG DSRGPVLFCQPRQGFNNRYFNVFKFRSMYTDMSD
1400
1401
ACCTTGACGCCGCGCGCCAGACATCGCGCACCGACCCGCG LDAARQTSRTDPRVTRIGRWIBRLSIDELPQLF
1500
1501
CAACGTGCTGCGTGGCGCGCT~~C~CCACGC NVLRGEMSLVGPRPHAPQTRAGGQLLHEAMEEY +uroyl GTCGCCCGCCACCGCGTGCAGCCCGOTATTACCGGCTGGG VARHRVQPGITGWAOINGSRGELVTRDDLCRRVV
1600
1001 1101 1201
O=ZVl--)
1601 1701
TGCTGGATCTGGAATACATTCGCGCA'IY;GTCGATCTGGGCGTTCTG LDLEYIRAWSIWLDIKI
1700 1800 IFLTIKREIFSRNAF*
1801
Fig. 1. Nucleotide and deduced amino acid sequence of uceA gene from A. .r~Vinum. Potential promoter (-35 and - 10 regions) and ribosome binding sites are marked. The positions of degenerate primers exoy I and exoy2 are indicated. The sequence has been deposited in the EMBL databank under the Accession number X93 149.
A.M. Grifln et al./ FEMS Microbiology
instructions. Taq polymerase was purchased from Perkin-Elmer, Norwalk, CT, USA. Standard molecular cloning, transformation and electrophoresis techniques were used as described [9]. DNA sequencing was performed on plasmid DNA with the ABI 373A automated sequencer. PCR amplifications were carried out in 50 ~1 reaction containing 1 pg genomic DNA, 100 pmol each primer, 2.5 U Taq polymerase, 1.5 mM MgCl,, 50 mM KCl, 10 mM Tris . HCl, pH 8.3, 0.001% gelatin and 5 mM each dNTP. DNA for use as probes was labelled using ECL Labelling System (Amersham, Bucks, UK). Degenerate PCR primers were designed with the aid of the computer
Letters 137 (1996) 115-121
117
program OLIGO (National Biosciences, Inc., Plymouth, MN). Sequence data were analyzed on a VAX 4600 using the computer analysis package GCG [ 101, CLUSTAL [l 11, FASTA and TFASTA [121.
3. Results and discussion 3.1. Cloning the aceA gene from A. xyiinum Genes encoding glycosyl-phosphate transferases catalyzing the first step in polysaccharide biosynthe-
1 ace4
aceA
aceA
aceA
ace4 SlrmD
ace4
aceA
aceA srnno
60 MGIISIiSIVQCCIXWgAGP~LS-SLPMIP MLLAD---LSSATYTPSSPRLLSKYSAA-----ADLVLRVF-----DLIMwASGL--IA l * .**** l * . . . . . . .* **. 61 120 PJuI-rrARGVDDSFSYRHPVL PQWVLSDAFCVCLAWACI?WJ~PJVPRIYarPDELSGALLLAN PYRVAI--AlTLLYSVICPALPPL-YRSWR----GRGLUELWLGG SAA** ** . l . .** . . .* ** l . .*. . .*. 121 180 IMAAGAFFLFPKNVPLLDIPD-ITKVSVQIRYL--LP ~VMLSWPFGVT AFG-GVFALPAVHAL I FLNHL-RTQGVD l l ** . . l l l * l * . .. . . .. . .. .. .. . .. 181 240 WMZ4EWLTFVVAI~ILI.W~ IIGSGSEA’K@M’RIWI’RMQ sRNpwvE;MNMv______-_________-___c;yFR VQRW-----WGLRHPWKISHYL l l * . . . . . . *. . * . . 241 300 RMFRLvvVLWBEDH IDAVIICFPP~~ TPYDLAVAEQRQGLPClGDPDFLI~ SLPLG-ERDHIKQLLQRUNYPI l . . ** * .**. . . . . . . l * .* . ..*. ..* ** 301 360 DVYVVPS PFSLWLQm---M NVIUI~SAEQIGSVPVINL-~DKILAVIALWL l l l . * . ** * . . *. . *. . . . . .* . . * .*. * . . * 361 420 APLLTLVALUKLDSRGPVLFCQPRQG~SRTDPR WPLML7UAVGVKMSSPGPVFPRQRFC+Z LIQQA_ * * . l ***** *480l .*.. **. * *iI*.* l l .,;l **. . . . .*... 421 . .A. .+I WI--f vrRIGRwIRRLsIDEuQ~vG PRPWLPQTRAGGQLLHEAME ITRFGsFLRRSSLDELPQIFIVG PRPWULQHNPHYEKL---INH!MQm ***** ,** * *******$I** * ** ******* . * . . * . . . t . l * *. 539 e+ PGIlGwAQIN LCRRWLDLEYIRAh’SIWLDIKIIFLTIKREIFSFNAF PGIVW IQYDLDYIRRWSLWLDIRIIVLTAVRVLGQKTAY l*t**tt**** *** ***t** *******.** ** * . . .l . . *.
Fig. 2. Alignment of the AceA sequence from A. xylinum with the GumD sequence from X. campestris. The positions of degenerate primers exoyl and exoy2, and a third conserved region are indicated, the confirming region is indicated (A). An asterisk indicates identity, a dot indicates a conservative replacement.
sis in several Gram-negative organisms were identified in the databanks. A multiple alignment of the amino acid sequences of KumD from Xcmthomorms cumpestris [ 131, exoY from Rhiwbium meliloti [ 141. r.xoY from Rhi:obium strain NGR 234 [IS]. p.ss2 from R. le~L~mino.sLIrllrn[ 161 and tfbP from S~lt~ot~elk typhimurium [ 171 was used to identify regions conserved between all genes. Degenerate primers were designed from these conserved regions taking into account information on Acetobacter codon usage (Figs. 1 and 2). The sequence of the exoyl primer (a 29 mer) was as follows: S-CGTCGT-TCS-WMS-CTK-GAY-GAR-CTK-CCS-CA-J and the sequence of exoy2 (a 30 mer) was: S’-GTTMAC-YTG-SGC-CCA-SCC-SGT-RAT-SCC-SGG3’. These primers were used in a PCR reaction to amplify a 180 bp fragment from A. qlirzum total genomic DNA. The translated sequence of this PCR product contained conserved region ‘A’ indicated in Fig. 2. The PCR product was labelled with tluoreacein and used to screen a lambda library of genomic DNA. One positive lambda clone was selected and subcloning its insert DNA (8.42 kb) into the Not1 site of pBluescript generated a plasmid clone named pAG1.
The sequence of a 1.9 kb region of pAG1 was determined (Fig. I). Computer analysis of this sequence data revealed the presence of an open reading
frame (ORF) that showed strong conformity with the Acetobucter codon usage table (not shown). The predicted 532 amino acid sequence of this ORF was compared to the translated (all six reading frames) DNA sequences in the EMBL and GenBank databanks using the program TFASTA. Strong homology was found with the translated gumD gene (Fig. 2) and with genes involved in polysaccharide biosynthesis in several other organisms (Table I). These genes have been postulated to encode the glycosylphosphate transferase catalyzing the first step ot polysaccharide biosynthesis in their respective hosts [ 14-231. For instance. in in vitro experiments, gumD and c.roY mutants did not accumulate lipid-linked biosynthetic intermediates. This indicated that these genes encoded functions involved in the first step in polysaccharide biosynthesis [ l&19]. Jiang et al. demonstrated that the rfbP gene encoded the galactosyl transferase likely to catalyze the first step of O-antigen biosynthesis [ 171. Thus it is likely that rrcrA encodes the glucose- 1-phosphate transferaae catalyzing the first step of the acetan biosynthetic pathway. The hydrophobicity plots of AceA. GumD and RfbP exhibited nearly identical structures except that AceA had four. not five, transmembrane domains in the amino two thirds of the protein (A. Griffin, unpublished data). Such hydrophobic regions are likely to constitute membrane embedded domains. ExoY and Pss2 are small proteins (approximately 37% of AceA) in which these hydrophobic anchor-
Table I Percentage similarity
and identity between the translated trwd gene from il.
\-v/irrw~ and the amino acid vqurnces
of gene> from other-
organism5 Gene
% Similarity
% Identity
Acceaion
Reference
55.32 (61.73)
39.36 (31.70)
u225 I I
[I31
52.03 (5X.70)
3
X.56703
1171 [20]
S6.X
B Similarity
I
(60.60)
22 (36.15)
77.7
I (36.01)
X7792 1
56.22 (67.98)
30.68 (43.42)
D2 1242
61.6.5
38.84
Q0273
hl .YO
10.00
I so33I I
66.X.3
SO.3
PI4186
ss. I3 (59.4.5)
26.62 (34.45)
s34977
52.65 (57.79)
25. I7 (36.23)
uo92.19
and % identity hcures were calculated uaing the KC;
program Beatit.
[211 [I-L,ZY] [l61
[ISI [Xl [231
Value\ m parentheses we the % similarity
and %
identity score.\obtained by comparison of the carboxy terminal 230 amino acids of each pair of translated sequences. The eroY and p.s.t2 genes only contain 226 and 200 ita respectively.
A.M. Grifln
et ul./
FEMS Microbiology
ing domains are missing. These proteins are known to be associated with the cell surface [ 14,211 therefore the anchoring function may be provided by hydrophobic residues at the N-terminus [14]. A periplasmic protein, encoded by exoF, is also required for the first step of polysaccharide biosynthesis in R. meliloti [14,24]. It has recently been postulated that rjbP may have arisen from a fusion of ancestral genes, bringing together a transferase activity and an unknown function provided by a hydrophobic protein [25]. Thus it is tempting to speculate that the hydrophobic amino regions of AceA, GumD and RfbP provide functions other than a sugar transferase activity. The carboxy terminal one third of RfbP [ 171 and GumD [ 131 are presumed to contain the catalytic domain involved in the transfer of a sugar-phosphate unit to the lipid carrier. We compared the carboxy terminal 230 amino acid region of translated genes with each other and found that the similarity and identity scores were much higher than those obtained from whole genes (Table 1). The identity score between AceA and GumD increased from 29% to 41.7% in this region, while that between AceA and ORF 14 increased from 30.68% to 43.42%. This type of analysis made comparisons to the ExoY and Pss2 sequences more meaningful, since these contain only this putative catalytic domain. We were surprised to find that AceA had a higher identity to ExoY from Rhizobium strain NGR 234 than to GumD from X. campestris. As mentioned previously, AceA and GumD are involved in the production of acetan and xanthan respectively; the biochemical pathways for these polymers are very similar, with the first four steps being identical. Analysis of an evolutionary tree depicting the relationship between AceA and its homologues showed that GumD was the first phospho-glucosyltransferase to evolve from the ancestral gene (Fig. 3). Divergence of AceA and Pss2 is a more recent event. As expected, this tree divides the glycosylphosphate transferases broadly into two groups: those that transfer galactose-phosphate to the lipid carrier (ExoY, RfbP, AmsG, CspD and CpsE) and those that transfer glucose-phosphate (GumD, AceA, Pss2 and ORF 14). The galactosyl transferases can be further divided into Gram-positive and Gram-negative transferases.
Letters
119
137 flYY61 115-121
exoY tm
-I+
exoY
n
aceA
pss
2
Fig. 3. Evolutionary relationships between the ExoY from R. (tm) and Rhizobium strain NCR 234 (n), RtbP from S. wphimurium, AmsG from E. amykwara, 5’ half of CpsD from S. agalactiae, CpsE from S. pneumoniae, AceA from A. x_ylinum, Pss2 from R. leguminosarum, ORF 14 from K. pneumoniae and GumD from X. campestris. The phylogenetic tree was constructed using the GCG programs Distance and GrowTree using the Kimura Proteins and UMPGA methods respectively. The program makes the assumption that the rate of gene substitution is constant and the distance measure is linear with evolutionary time. Horizontal branches are proportional to the distance between the sequences. meliloti
The genes for polysaccharide biosynthesis are clustered in the majority of organisms [26], therefore using aceA as a probe, it should be relatively easy to clone and nucleotide sequence the remainder of the acetan cluster. An important point to note here is that, while the first step in polysaccharide biosynthesis (i.e. transfer of a sugar-phosphate unit to a lipid carrier) is conserved among a large number of organisms, steps catalyzed by other transferases will be unique to each host. These will be determined by the particular polysaccharide synthesized by the host; sugar transferases are specific for the identity and
120
A.M. Gr#in
et (II./
FEMS Microbiology
anomeric configuration of the sugar unit, and linkage between the sugar units in the polysaccharide. Thus strategies which target the first step in polysaccharide biosynthesis could be used to find gene clusters involved in polysaccharide biosynthesis in a wide range of organisms. Likewise, strategies for gene inactivation that target the first step in the pathway could be used to switch off polysaccharide production. In the case of A. xyfinum, inactivation of the aceA gene could be used to further channel cellular UDP-glucose into cellulose production thereby increasing the yield of cellulose produced by A. xylinum and making the goal of using bacteria1 cellulose for industrial processes more attainable [27,28].
Acknowledgements This work was funded by the Biotechnology Biological Sciences Research Council.
and
References [II
Leigh, J.L. and Coplin, D.L. (1992) Exopolysaccharidea in plant-bacterial interactions. Annu. Rev. Micobiol. 46, 307346. 121Colquhoun, I.J., Defemez, M. and Morris, V.J. (1995) NMR studies of acetan and the related bacterial polysaccharide, CR I /4, produced by a mutant strain of Acetobacter xylinum. Carbohydrate Res. 269, 3 19-33 I. I.W. (1990) Biotechnology of Microbial Ex131 Sutherland, opolysaccharides. Cambridge University Press, Cambridge. 141Brown, R.M. Jr. (1989) Bacterial cellulose. In: Cellulose: Structural and Functional Aspects (Kennedy, J.F., Phillips, G.O. and Williams, P.A., Eds.), pp. 145-151. Ellis Horwood, Chichester. 151Jansson, P.E., Kenne, L. and Lindberg. 8. (1975) Structure of the extracellular polysaccharide from Xanthomonas campestris. Carbohydrate Res. 45. 275-282. [61 de Iannino, N.I., Couso, R.O. and Dankert, M.A. (1988) Lipid-linked intermediates and the synthesis of acetan in Acetobacfer qlinum. J. Gen. Microbial. 134, 173 I - 1736. 171 MacCormick, C.A., Harris, J.E., Gunning, A.P. and Morris, V.J. (1993) Characterization of a variant of the polysaccharide acetan produced by a mutant of Acetobacter xylinum strain CR1 /4. J. Appl. Bacterial. 74, 196-199. [81 Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [91 Sambrook, J., Fritsch, E.F. and Maniatis, T. (I 989). Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Letters 137 (19961 115-121
1101 Devereux, J., Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the Vax. Nucleic Acids Res. 12. 216-223. [Ill Higgins, D.G. (1994) CLUSTAL V: Multiple alignment of DNA and protein sequences. In: Computer Analysis of Sequence Data, Part 2 (Griffin, A.M. and Griffin, H.G., Eds.), pp. 307-318. Humana Press, Totowa, NJ. [121 Pearson, W.R. (1994) Using the FASTA program to search protein and DNA sequence databases. In: Computer Analysis of Sequence Data, Part 1 (Griffin, A.M. and Griffin, H.G., Eds.), pp. 307-33 I. Humana Press, Totowa. NJ. [131 Capage, M.A., Doherty, D.H.. Betlach, M.R. and Vanderslice, R.W. (1987) Recombinant-DNA mediated production of xanthan gum. Int. Pat. Pub. Number WO87/05938. [I41 Muller, P., Keller, M., Weng, W.M., Quandt, J., Arnold, W. and Puhler, A. (1993) Genetic analysis of the Rhizobium meliloti ExoYFQ operon - ExoY is homologous to the sugar transferases and ExoQ represents a transmembrane protein, Mol. Plant-Micro. Inter. 6, 55-65. [151 Gray, J.X., Djordjevic, M.A. and Rolfe, B.G. (1990) Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotype. J. Bacterial. 172, 193-203. 1161Borthakur, D., Barker, R.F., Rossen, L. and Johnston, A.W.B. (1988) Analysis of pss genes of Rhizobium leguminosurum required for exopolysaccharide synthesis and nodulation of peas: Their primary structure and their interaction with psi and other nodulation genes. Mol. Gen. Genet. 2 13, 155- 162. [I71 Jiang, X.-M., Neal, B., Santiago, F., Lee, S.J., Romana, L.K. and Reeves, P.R. (1991) Structure and sequence of the+ (0 antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol. Microbial. 5, 695-713. 1181Vanderslice, R.W., Doherty, D.H., Capage, M.A., Betlach, M.R., Hassler, R.A., Henderson, N.M., Ryan-Graniero. J. and Tecklenburg, M. (1990) Genetic engineering of polysaccharide structure in Xanthomonas campestris. In: Biomedical and Biotechnological Advances in Industrial Polysaccharides (Cresenzi, V., Dea, I.C.M., Paoletti, S., Stivala, S.S. and Sutherland, I.W., Eds.), pp. 145-156. Gordon and Breach Science, New York. [I91 Reuber, T.L. and Walker, G.C. (1993) Biosynthesis of succinoglycan a symbiotically important exopolysaccharide of Rhizobium
meliloti.
Cell 74, 269-280.
1201Bugert, P. and Geider, K. (1995) Molecular
analysis of the for exopolysaccharide synthesis of Erwinia amylowra. Mol. Microbial. 15, 917-933. R., Nagatsuka, T., Ito, H.. 1211Arakawa, R., Wacharotayankun, Kato, N. and Ohta, M. (1995) Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J. Bacterial. 177, 1788-1796. I221 Rubens, C.E., Heggen, L.M., Haft, R.F. and Wessels, M.R. (1993) Identification of cpsD, a gene essential for Type III capsule expression in Group B Streptococci. Mol. Microbial. 8, 843-855. 1231Guidolin, A., Morona, J.K., Morona, R., Hansman, D. and Paton, J.C. (1994) Nucleotide-sequence analysis of genes ams operon required
A.M. Grifin
et al. / FEMS Microbic;‘logy Letters 137 (19961 115-121
essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. Infect. Immun. 62,5384-5396. [24] Leigh, J.A. and Walker, G.C. (1994) Exopolysaccharides of Rhizobium - synthesis, regulation and symbiotic function. Trends Genet. 10, 63-67. [25] Schnaitman, C.A. and Klena, J.D. (1993) Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol. Rev. 57, 655-682. [26] Coplin, D.L. and Cook, D. (1990) Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol. Plant-Micro. Inter. 5, 27 l-279.
121
[27] Oikawa, T., Ohtori, T. and Ameyama, M. (1995) Production of cellulose from D-mannitol by Acetobacter xylinum KU- 1. Biosci. Biotech. Biochem. 59, 331-332. 1281 Ross, P., Mayer, R. and Benziman, M. (1991) Cellulose biosynthesis and function in bacteria. Microbial. Rev. 55, 35-58. [29] Reed, J.W., Capage, M. and Walker, CC. (1991) Rhizobium meliloti exoG and exoJ mutations affect the ExoX-ExoY system for modulation of exopolysaccharide production. J. Bacterial. 173, 3776-3788.
ELSEVIER
FEMS Microbiology
Letters I37 ( 1996)
I IS I2 I
Identification, cloning and sequencing the aceA gene involved in acetan biosynthesis in Acetobacter xylinum *, Victor
Annette M. Griffin Received 23 November
J. Morris, Michael J. Gasson
1995; revised 22 January
1996; accepted 23 January
1996
Abstract The aceA gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of gumD from Xanthomonas campestris. Therefore aceA is likely to encode the phosphate-prenyl glucose l-phosphate transferase catalyzing the first step in acetan biosynthesis in A. xylinum. Kqwwdc:
Gene: Acetan; Cloning:
DNA sequencing;
Exopolysaccharide;
1. Introduction Many microorganisms have the ability to synthesize extracellular polysaccharides (EPS) and excrete them from the cell, as either soluble or insoluble polymers. Such polymers confer selective advantages on the host; for example, allowing adherence to inert surfaces, protection from desiccation and other environmental stresses, antigenic variation of pathogenic strains, and masking immune recognition. Consequently bacterial EPS provide important model systems for the study of molecular assembly and secretion, gene regulation, cell-cell interactions, symbiosis, pathogenesis, and the relationship between polysaccharide structure and function [1,2]. In addition, microbial polymers, such as xanthan, are widely used in the food and other industries as gelling,
* Corresponding author. Tel.: +44 (1603) 255 000 or 255 354; Fax: + 44 (I 603) 507 723; E-mail: [email protected] 037% 1097/96/$15.00 0 1996 Federation PII SO378- 1097(96)00049-3
of European
Microbiological
Acetohacter
xylinum
thickening, texturizing, suspending and encapsulating agents [3]. In recent years there has been considerable interest in developing novel microbial polysaccharides for use in industry and one of the long-term aims of these studies is to genetically engineer new polymers with altered structures that confer improved and novel properties. Acetobacter xylinum is used in the production of a sweet confectionery (Natal enjoyed in many areas around the world [4]. This organism produces two different extracellular polysaccharides: cellulose and acetan. Acetan is an acidic polysaccharide which is structurally related to xanthan [5]. In order to engineer novel acetan based polysaccharides it is essential to genetically characterize the pathway for biosynthesis. Although the biochemical pathway for acetan biosynthesis has been elucidated [6], nothing is known of the genetics of this process. This paper describes our strategy for the identification, cloning and sequencing of the A. xylinum aceA gene predicted to encode the first step in acetan biosynthesis. Societies. All rights reserved
2. Materials
and methods
2. I. Media and reagents The Cl strain of A. xylinum was grown in AJ medium at 30°C as described previously 171. Stains of Escherichia coli strains were grown in L-broth or on L-agar [8], or NZYCM media [9] at 37°C. Ampicillin was added to a concentration of 100 pg ml-’ when required. Strains for plating lambda phage were grown in L-broth containing 0.2% maltose and 10 mM MgSO, at 30°C as described [9]. Lambda were stored in SM buffer [9] at 4°C.
@age 1
2.2. Bacterial strains and plusmids A library of A. xylinum strain Cl was constructed in lambda DASH II (Stratagene, Cambridge, UK). E. coli XLI-Blue P2 (Stratagene) was used as the host for phage lambda and strain TGl [9] was used as a host for plasmids. 2.3. DNA manipulations Restriction enzymes and DNA modifying enzymes were purchased from Gibco BRL, Paisley, UK and were used according to the manufacturer’s
35 -TTTCATGAAT
TGTCCGTGGTTTTGTCTTTTCTGGCCTCATATCGTTACGA
'ATTGGGAAATTC
100
101
CTTCCATGTATTGATGTCGTTATTTTTCGCCTCCAAA
200
201
AAATGGGGATTATTTCGCACTCGATn;TGCAGTGCGGA MGIISHSIVQCGKWQAGPARRQVVFRVHRRCFK
300
301
AATGAGCGAGGCACTCAGGCACTTGAAGAATGGCGAAACG~CGCTACCAT~CGGTCTCGA~GCATTCCC~~GCATCC~TAC~GCG~ MSEALRHLKNGETDLSSLPSGLDSIPRRIHTAR
400
401
GGCGTGGATGACAGCTTTTCCTACCGGCATCCTGTCCTGC GVDDSFSYRHPVLPQVVVLSDAFCVCLAVVACIA
500
501
CCTGGCAGAGGATATACGATACCCCCGATGAACTGTCTGGT WQRIYDTPDELSGALLLANIMAAGAFFLFPKNV
600
601
TCCCCTCCTCGACATTCCCGCATAACCAAGGTTTCTGTC PLLDIPDITKVSVQIRYLLPPVAFGEVVFCTVL
700
701
GTCATGCTGTCATGGCCGTTTGGCGTGACGGTGCGCATGG VMLSWPFGVTVRMGMEWLTFVVAILFVERCVGTY
800
801 901
GCGCATGCAGCGCATGTTCCGCCTCGTCGGGACATTTGAT RMQRMFRLVGT FDDQGGDSDGTVEELVLRARED CATATCGATGCGGTCATCATATGCTTTCCCCCCGCGTGCG HIDAVIICFPPACGQQHVMDVLWRLRGVLADVYV
1100
1301
GACTCCCGCGGGCCGGTGCTGTTCTGCCAGCCCCGGCAGG DSRGPVLFCQPRQGFNNRYFNVFKFRSMYTDMSD
1400
1401
ACCTTGACGCCGCGCGCCAGACATCGCGCACCGACCCGCG LDAARQTSRTDPRVTRIGRWIBRLSIDELPQLF
1500
1501
CAACGTGCTGCGTGGCGCGCT~~C~CCACGC NVLRGEMSLVGPRPHAPQTRAGGQLLHEAMEEY +uroyl GTCGCCCGCCACCGCGTGCAGCCCGOTATTACCGGCTGGG VARHRVQPGITGWAOINGSRGELVTRDDLCRRVV
1600
1001 1101 1201
O=ZVl--)
1601 1701
TGCTGGATCTGGAATACATTCGCGCA'IY;GTCGATCTGGGCGTTCTG LDLEYIRAWSIWLDIKI
1700 1800 IFLTIKREIFSRNAF*
1801
Fig. 1. Nucleotide and deduced amino acid sequence of uceA gene from A. .r~Vinum. Potential promoter (-35 and - 10 regions) and ribosome binding sites are marked. The positions of degenerate primers exoy I and exoy2 are indicated. The sequence has been deposited in the EMBL databank under the Accession number X93 149.
A.M. Grifln et al./ FEMS Microbiology
instructions. Taq polymerase was purchased from Perkin-Elmer, Norwalk, CT, USA. Standard molecular cloning, transformation and electrophoresis techniques were used as described [9]. DNA sequencing was performed on plasmid DNA with the ABI 373A automated sequencer. PCR amplifications were carried out in 50 ~1 reaction containing 1 pg genomic DNA, 100 pmol each primer, 2.5 U Taq polymerase, 1.5 mM MgCl,, 50 mM KCl, 10 mM Tris . HCl, pH 8.3, 0.001% gelatin and 5 mM each dNTP. DNA for use as probes was labelled using ECL Labelling System (Amersham, Bucks, UK). Degenerate PCR primers were designed with the aid of the computer
Letters 137 (1996) 115-121
117
program OLIGO (National Biosciences, Inc., Plymouth, MN). Sequence data were analyzed on a VAX 4600 using the computer analysis package GCG [ 101, CLUSTAL [l 11, FASTA and TFASTA [121.
3. Results and discussion 3.1. Cloning the aceA gene from A. xyiinum Genes encoding glycosyl-phosphate transferases catalyzing the first step in polysaccharide biosynthe-
1 ace4
aceA
aceA
aceA
ace4 SlrmD
ace4
aceA
aceA srnno
60 MGIISIiSIVQCCIXWgAGP~LS-SLPMIP MLLAD---LSSATYTPSSPRLLSKYSAA-----ADLVLRVF-----DLIMwASGL--IA l * .**** l * . . . . . . .* **. 61 120 PJuI-rrARGVDDSFSYRHPVL PQWVLSDAFCVCLAWACI?WJ~PJVPRIYarPDELSGALLLAN PYRVAI--AlTLLYSVICPALPPL-YRSWR----GRGLUELWLGG SAA** ** . l . .** . . .* ** l . .*. . .*. 121 180 IMAAGAFFLFPKNVPLLDIPD-ITKVSVQIRYL--LP ~VMLSWPFGVT AFG-GVFALPAVHAL I FLNHL-RTQGVD l l ** . . l l l * l * . .. . . .. . .. .. .. . .. 181 240 WMZ4EWLTFVVAI~ILI.W~ IIGSGSEA’K@M’RIWI’RMQ sRNpwvE;MNMv______-_________-___c;yFR VQRW-----WGLRHPWKISHYL l l * . . . . . . *. . * . . 241 300 RMFRLvvVLWBEDH IDAVIICFPP~~ TPYDLAVAEQRQGLPClGDPDFLI~ SLPLG-ERDHIKQLLQRUNYPI l . . ** * .**. . . . . . . l * .* . ..*. ..* ** 301 360 DVYVVPS PFSLWLQm---M NVIUI~SAEQIGSVPVINL-~DKILAVIALWL l l l . * . ** * . . *. . *. . . . . .* . . * .*. * . . * 361 420 APLLTLVALUKLDSRGPVLFCQPRQG~SRTDPR WPLML7UAVGVKMSSPGPVFPRQRFC+Z LIQQA_ * * . l ***** *480l .*.. **. * *iI*.* l l .,;l **. . . . .*... 421 . .A. .+I WI--f vrRIGRwIRRLsIDEuQ~vG PRPWLPQTRAGGQLLHEAME ITRFGsFLRRSSLDELPQIFIVG PRPWULQHNPHYEKL---INH!MQm ***** ,** * *******$I** * ** ******* . * . . * . . . t . l * *. 539 e+ PGIlGwAQIN LCRRWLDLEYIRAh’SIWLDIKIIFLTIKREIFSFNAF PGIVW IQYDLDYIRRWSLWLDIRIIVLTAVRVLGQKTAY l*t**tt**** *** ***t** *******.** ** * . . .l . . *.
Fig. 2. Alignment of the AceA sequence from A. xylinum with the GumD sequence from X. campestris. The positions of degenerate primers exoyl and exoy2, and a third conserved region are indicated, the confirming region is indicated (A). An asterisk indicates identity, a dot indicates a conservative replacement.
sis in several Gram-negative organisms were identified in the databanks. A multiple alignment of the amino acid sequences of KumD from Xcmthomorms cumpestris [ 131, exoY from Rhiwbium meliloti [ 141. r.xoY from Rhi:obium strain NGR 234 [IS]. p.ss2 from R. le~L~mino.sLIrllrn[ 161 and tfbP from S~lt~ot~elk typhimurium [ 171 was used to identify regions conserved between all genes. Degenerate primers were designed from these conserved regions taking into account information on Acetobacter codon usage (Figs. 1 and 2). The sequence of the exoyl primer (a 29 mer) was as follows: S-CGTCGT-TCS-WMS-CTK-GAY-GAR-CTK-CCS-CA-J and the sequence of exoy2 (a 30 mer) was: S’-GTTMAC-YTG-SGC-CCA-SCC-SGT-RAT-SCC-SGG3’. These primers were used in a PCR reaction to amplify a 180 bp fragment from A. qlirzum total genomic DNA. The translated sequence of this PCR product contained conserved region ‘A’ indicated in Fig. 2. The PCR product was labelled with tluoreacein and used to screen a lambda library of genomic DNA. One positive lambda clone was selected and subcloning its insert DNA (8.42 kb) into the Not1 site of pBluescript generated a plasmid clone named pAG1.
The sequence of a 1.9 kb region of pAG1 was determined (Fig. I). Computer analysis of this sequence data revealed the presence of an open reading
frame (ORF) that showed strong conformity with the Acetobucter codon usage table (not shown). The predicted 532 amino acid sequence of this ORF was compared to the translated (all six reading frames) DNA sequences in the EMBL and GenBank databanks using the program TFASTA. Strong homology was found with the translated gumD gene (Fig. 2) and with genes involved in polysaccharide biosynthesis in several other organisms (Table I). These genes have been postulated to encode the glycosylphosphate transferase catalyzing the first step ot polysaccharide biosynthesis in their respective hosts [ 14-231. For instance. in in vitro experiments, gumD and c.roY mutants did not accumulate lipid-linked biosynthetic intermediates. This indicated that these genes encoded functions involved in the first step in polysaccharide biosynthesis [ l&19]. Jiang et al. demonstrated that the rfbP gene encoded the galactosyl transferase likely to catalyze the first step of O-antigen biosynthesis [ 171. Thus it is likely that rrcrA encodes the glucose- 1-phosphate transferaae catalyzing the first step of the acetan biosynthetic pathway. The hydrophobicity plots of AceA. GumD and RfbP exhibited nearly identical structures except that AceA had four. not five, transmembrane domains in the amino two thirds of the protein (A. Griffin, unpublished data). Such hydrophobic regions are likely to constitute membrane embedded domains. ExoY and Pss2 are small proteins (approximately 37% of AceA) in which these hydrophobic anchor-
Table I Percentage similarity
and identity between the translated trwd gene from il.
\-v/irrw~ and the amino acid vqurnces
of gene> from other-
organism5 Gene
% Similarity
% Identity
Acceaion
Reference
55.32 (61.73)
39.36 (31.70)
u225 I I
[I31
52.03 (5X.70)
3
X.56703
1171 [20]
S6.X
B Similarity
I
(60.60)
22 (36.15)
77.7
I (36.01)
X7792 1
56.22 (67.98)
30.68 (43.42)
D2 1242
61.6.5
38.84
Q0273
hl .YO
10.00
I so33I I
66.X.3
SO.3
PI4186
ss. I3 (59.4.5)
26.62 (34.45)
s34977
52.65 (57.79)
25. I7 (36.23)
uo92.19
and % identity hcures were calculated uaing the KC;
program Beatit.
[211 [I-L,ZY] [l61
[ISI [Xl [231
Value\ m parentheses we the % similarity
and %
identity score.\obtained by comparison of the carboxy terminal 230 amino acids of each pair of translated sequences. The eroY and p.s.t2 genes only contain 226 and 200 ita respectively.
A.M. Grifln
et ul./
FEMS Microbiology
ing domains are missing. These proteins are known to be associated with the cell surface [ 14,211 therefore the anchoring function may be provided by hydrophobic residues at the N-terminus [14]. A periplasmic protein, encoded by exoF, is also required for the first step of polysaccharide biosynthesis in R. meliloti [14,24]. It has recently been postulated that rjbP may have arisen from a fusion of ancestral genes, bringing together a transferase activity and an unknown function provided by a hydrophobic protein [25]. Thus it is tempting to speculate that the hydrophobic amino regions of AceA, GumD and RfbP provide functions other than a sugar transferase activity. The carboxy terminal one third of RfbP [ 171 and GumD [ 131 are presumed to contain the catalytic domain involved in the transfer of a sugar-phosphate unit to the lipid carrier. We compared the carboxy terminal 230 amino acid region of translated genes with each other and found that the similarity and identity scores were much higher than those obtained from whole genes (Table 1). The identity score between AceA and GumD increased from 29% to 41.7% in this region, while that between AceA and ORF 14 increased from 30.68% to 43.42%. This type of analysis made comparisons to the ExoY and Pss2 sequences more meaningful, since these contain only this putative catalytic domain. We were surprised to find that AceA had a higher identity to ExoY from Rhizobium strain NGR 234 than to GumD from X. campestris. As mentioned previously, AceA and GumD are involved in the production of acetan and xanthan respectively; the biochemical pathways for these polymers are very similar, with the first four steps being identical. Analysis of an evolutionary tree depicting the relationship between AceA and its homologues showed that GumD was the first phospho-glucosyltransferase to evolve from the ancestral gene (Fig. 3). Divergence of AceA and Pss2 is a more recent event. As expected, this tree divides the glycosylphosphate transferases broadly into two groups: those that transfer galactose-phosphate to the lipid carrier (ExoY, RfbP, AmsG, CspD and CpsE) and those that transfer glucose-phosphate (GumD, AceA, Pss2 and ORF 14). The galactosyl transferases can be further divided into Gram-positive and Gram-negative transferases.
Letters
119
137 flYY61 115-121
exoY tm
-I+
exoY
n
aceA
pss
2
Fig. 3. Evolutionary relationships between the ExoY from R. (tm) and Rhizobium strain NCR 234 (n), RtbP from S. wphimurium, AmsG from E. amykwara, 5’ half of CpsD from S. agalactiae, CpsE from S. pneumoniae, AceA from A. x_ylinum, Pss2 from R. leguminosarum, ORF 14 from K. pneumoniae and GumD from X. campestris. The phylogenetic tree was constructed using the GCG programs Distance and GrowTree using the Kimura Proteins and UMPGA methods respectively. The program makes the assumption that the rate of gene substitution is constant and the distance measure is linear with evolutionary time. Horizontal branches are proportional to the distance between the sequences. meliloti
The genes for polysaccharide biosynthesis are clustered in the majority of organisms [26], therefore using aceA as a probe, it should be relatively easy to clone and nucleotide sequence the remainder of the acetan cluster. An important point to note here is that, while the first step in polysaccharide biosynthesis (i.e. transfer of a sugar-phosphate unit to a lipid carrier) is conserved among a large number of organisms, steps catalyzed by other transferases will be unique to each host. These will be determined by the particular polysaccharide synthesized by the host; sugar transferases are specific for the identity and
120
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et (II./
FEMS Microbiology
anomeric configuration of the sugar unit, and linkage between the sugar units in the polysaccharide. Thus strategies which target the first step in polysaccharide biosynthesis could be used to find gene clusters involved in polysaccharide biosynthesis in a wide range of organisms. Likewise, strategies for gene inactivation that target the first step in the pathway could be used to switch off polysaccharide production. In the case of A. xyfinum, inactivation of the aceA gene could be used to further channel cellular UDP-glucose into cellulose production thereby increasing the yield of cellulose produced by A. xylinum and making the goal of using bacteria1 cellulose for industrial processes more attainable [27,28].
Acknowledgements This work was funded by the Biotechnology Biological Sciences Research Council.
and
References [II
Leigh, J.L. and Coplin, D.L. (1992) Exopolysaccharidea in plant-bacterial interactions. Annu. Rev. Micobiol. 46, 307346. 121Colquhoun, I.J., Defemez, M. and Morris, V.J. (1995) NMR studies of acetan and the related bacterial polysaccharide, CR I /4, produced by a mutant strain of Acetobacter xylinum. Carbohydrate Res. 269, 3 19-33 I. I.W. (1990) Biotechnology of Microbial Ex131 Sutherland, opolysaccharides. Cambridge University Press, Cambridge. 141Brown, R.M. Jr. (1989) Bacterial cellulose. In: Cellulose: Structural and Functional Aspects (Kennedy, J.F., Phillips, G.O. and Williams, P.A., Eds.), pp. 145-151. Ellis Horwood, Chichester. 151Jansson, P.E., Kenne, L. and Lindberg. 8. (1975) Structure of the extracellular polysaccharide from Xanthomonas campestris. Carbohydrate Res. 45. 275-282. [61 de Iannino, N.I., Couso, R.O. and Dankert, M.A. (1988) Lipid-linked intermediates and the synthesis of acetan in Acetobacfer qlinum. J. Gen. Microbial. 134, 173 I - 1736. 171 MacCormick, C.A., Harris, J.E., Gunning, A.P. and Morris, V.J. (1993) Characterization of a variant of the polysaccharide acetan produced by a mutant of Acetobacter xylinum strain CR1 /4. J. Appl. Bacterial. 74, 196-199. [81 Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [91 Sambrook, J., Fritsch, E.F. and Maniatis, T. (I 989). Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Letters 137 (19961 115-121
1101 Devereux, J., Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the Vax. Nucleic Acids Res. 12. 216-223. [Ill Higgins, D.G. (1994) CLUSTAL V: Multiple alignment of DNA and protein sequences. In: Computer Analysis of Sequence Data, Part 2 (Griffin, A.M. and Griffin, H.G., Eds.), pp. 307-318. Humana Press, Totowa, NJ. [121 Pearson, W.R. (1994) Using the FASTA program to search protein and DNA sequence databases. In: Computer Analysis of Sequence Data, Part 1 (Griffin, A.M. and Griffin, H.G., Eds.), pp. 307-33 I. Humana Press, Totowa. NJ. [131 Capage, M.A., Doherty, D.H.. Betlach, M.R. and Vanderslice, R.W. (1987) Recombinant-DNA mediated production of xanthan gum. Int. Pat. Pub. Number WO87/05938. [I41 Muller, P., Keller, M., Weng, W.M., Quandt, J., Arnold, W. and Puhler, A. (1993) Genetic analysis of the Rhizobium meliloti ExoYFQ operon - ExoY is homologous to the sugar transferases and ExoQ represents a transmembrane protein, Mol. Plant-Micro. Inter. 6, 55-65. [151 Gray, J.X., Djordjevic, M.A. and Rolfe, B.G. (1990) Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotype. J. Bacterial. 172, 193-203. 1161Borthakur, D., Barker, R.F., Rossen, L. and Johnston, A.W.B. (1988) Analysis of pss genes of Rhizobium leguminosurum required for exopolysaccharide synthesis and nodulation of peas: Their primary structure and their interaction with psi and other nodulation genes. Mol. Gen. Genet. 2 13, 155- 162. [I71 Jiang, X.-M., Neal, B., Santiago, F., Lee, S.J., Romana, L.K. and Reeves, P.R. (1991) Structure and sequence of the+ (0 antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol. Microbial. 5, 695-713. 1181Vanderslice, R.W., Doherty, D.H., Capage, M.A., Betlach, M.R., Hassler, R.A., Henderson, N.M., Ryan-Graniero. J. and Tecklenburg, M. (1990) Genetic engineering of polysaccharide structure in Xanthomonas campestris. In: Biomedical and Biotechnological Advances in Industrial Polysaccharides (Cresenzi, V., Dea, I.C.M., Paoletti, S., Stivala, S.S. and Sutherland, I.W., Eds.), pp. 145-156. Gordon and Breach Science, New York. [I91 Reuber, T.L. and Walker, G.C. (1993) Biosynthesis of succinoglycan a symbiotically important exopolysaccharide of Rhizobium
meliloti.
Cell 74, 269-280.
1201Bugert, P. and Geider, K. (1995) Molecular
analysis of the for exopolysaccharide synthesis of Erwinia amylowra. Mol. Microbial. 15, 917-933. R., Nagatsuka, T., Ito, H.. 1211Arakawa, R., Wacharotayankun, Kato, N. and Ohta, M. (1995) Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J. Bacterial. 177, 1788-1796. I221 Rubens, C.E., Heggen, L.M., Haft, R.F. and Wessels, M.R. (1993) Identification of cpsD, a gene essential for Type III capsule expression in Group B Streptococci. Mol. Microbial. 8, 843-855. 1231Guidolin, A., Morona, J.K., Morona, R., Hansman, D. and Paton, J.C. (1994) Nucleotide-sequence analysis of genes ams operon required
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et al. / FEMS Microbic;‘logy Letters 137 (19961 115-121
essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. Infect. Immun. 62,5384-5396. [24] Leigh, J.A. and Walker, G.C. (1994) Exopolysaccharides of Rhizobium - synthesis, regulation and symbiotic function. Trends Genet. 10, 63-67. [25] Schnaitman, C.A. and Klena, J.D. (1993) Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol. Rev. 57, 655-682. [26] Coplin, D.L. and Cook, D. (1990) Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol. Plant-Micro. Inter. 5, 27 l-279.
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[27] Oikawa, T., Ohtori, T. and Ameyama, M. (1995) Production of cellulose from D-mannitol by Acetobacter xylinum KU- 1. Biosci. Biotech. Biochem. 59, 331-332. 1281 Ross, P., Mayer, R. and Benziman, M. (1991) Cellulose biosynthesis and function in bacteria. Microbial. Rev. 55, 35-58. [29] Reed, J.W., Capage, M. and Walker, CC. (1991) Rhizobium meliloti exoG and exoJ mutations affect the ExoX-ExoY system for modulation of exopolysaccharide production. J. Bacterial. 173, 3776-3788.