CATECHOL 2,3-DIOXYGENASE (C230) ACTIVITY ASSAY

2.2.23.1. AGAR PLATE DETECTION METHOD

Functional expression of the xvlE gene was detected by spraying antibiotic selection plates with an aqueous solu­ tion of 0.5 M catechol. C o l onies that express the gene become yellow due to the conversion by catechol 2,3-diox­ ygenase to 2-hydroxymucomic semialdehyde (Zukowski et al .. 1983).

2.2.23.2. SPECTROPHOTOMETRIC ASSAY

For enzymatic assays, the levels of C230 produced were measured spectrophotometrically (Sala-Trepat & Evans, 1971) from sonic extracts prepared essentially as described by Zukowski et a l .. 1983; Zukowski & Miller, 1986.

Cell cultures were grown overnight in 10 ml of appropriate broth and harvested by centrifugation (Sorval RC5B, SS24 rotor, 6,000 r.p.m., 10 min). The cells were washed in 5 ml 20 mM phosphate buffer, pH 7.2, buffer, recentrifuged as above, and resuspended in 1 ml 100 mM phosphate buffer, 10% acetone (v/v), pH 7.5. The cell suspensions were cooled on ice and then disrupted by three thirty sec sonications (MSE Soniprep 150) with 30 sec cooling intervals between bursts. The extracts w ere then c e n t r i f u g e d (Sorval RC5B, SHTM rotor, 12,000 r.p.m., 4°C, 15 min) to remove cellular debris. The supernatant ("extract") was decanted to a fresh tube and kep t on ice. C230 a c t i v i t y was de t e r m i n e d by following the increase in absorbance at 375 nm due to the accumulation of 2-hydroxymuconic semialdehyde; cuvettes

contained in a total volume of 3 ml: 250 fiM of phosphate

buffer, pH 8.0, 0.2 /*M of catechol and extract (between 1 111 and 100 /il depending on the amount of activity present. All measurements were carried out at 30°C. One unit of enzyme was defined as that amount which oxidised one micro­ mole of catechol per min at 30°C. The molar extinction coefficient of the product used was that of Nozaki (1970), being 4.4 X 104 ; one unit of enzyme activity corresponded

to an increase in absorbance of 14.8 min- 1 . Specific activ­ ity was expressed as units per milligram of protein.

2.2.24. CARBOXYPEPTIDASE G2 PLATE ASSAY

Ej_coli cells harbouring a functional carboxypeptidase G2

gene were detected by the Minimal media/folate agar plate method described by Minton & Clarke (1985). This method is reliant on the observation that a functional crboxypepti- dase G 2 gene enables E. coli strains to utilise folate as a sole carbon source with the production of a bright yellow p r o d u c t . C e l l s w e r e s i m p l y p l a t e d o n t o m i n i m a l medium/folate aga r plates and i ncubated for 48 hour at 3 7 ° C , by which time those carbox y p e p t i d a s e G 2 positive c o l o n i e s p r e s e n t wer e e a s i l y d e t e c t e d by t h e i r bright yellow colour.

2.2.25. PROTEIN DETERMINATION

The Coomassie Brilliant blue G250 binding assay was used to

estimate protein levels in sonic extracts. 0 . 2 ml of dye

reagent concentrate (Biorad Protein Assay Bulletin 1069) was added to 0.8 ml of sample, co n t a i n i n g 2 to 25 /ig of protein. After gentle mixing, and a period of 5 to 60 min at roo m temperature, the abs o r b a n c e at 595 nm was read a gainst a reagent blank. The p r o t e i n concentration was determined from a calibration curve using BSA as standard.

till__ IWTRQPgCTIQE

As no suitably marked plasmids had been identified from any

saccharolytic Clostridia, it was decided to undertake the in vitro construction of a plasmid vector by combining the replicative machinery of a cryptic plasmid with a suitable

genetic marker known to be phenotypically expressed in the intended clostridial host (e.g., an antibiotic resistance gene). The availability of such a vector should then facil­ itate the development of a transformation procedure.

At this time the most extensively characterised small

plasmids, derived from saccharolytic Clostridia, were the cryptic plasmids pCBlOl (5.6 kb), pCB102 (7.4 kb), and pCB103 (6.2 kb) isolated from C. butvricum (strains NCIB 7423 and NCTC 6084). First d e scribed by Minton & Morris (1981), these same plasmids were also isolated and partial­

ly characterised by Luczak et a l . (1985). Both groups of researchers simultaneously published restriction endonu­ clease maps for all three plasmids (Collins et a l .. 1985; Luczak et a l ., 1985).

Additional studies were directed towards the identification of the minimal replicón of these plasmids. In the absence of a reliable transformation procedure for saccharolytic

Clostridia, studies were performed in B. subtilis. Various

restriction endonuclease fragments derived from pCBlOl and PCB102 (Collins et a l .. 1985) and pCB103 (Minton & Clarke,

unpublished data) were cloned into Gram-positive replica­

tion-deficient plasmid vectors (i.e., "replicón cloning

vectors") and the ability of the resultant chimaeras to

replicate in B. subtilis tested. Collins et a l . (1985)

identified a 3.3 kb S a u 3A fragment of pCBlOl which con­ ferred on the replicón cloning vect o r used (pJABl) the

ability to autonomously replicate in B.subtilis. They also

identified a 2.0 kb fragment of pCB102 which, upon appro­ priate selection, caused the chimaeric construction with pJABl to integrate into the B. subtilis chromosome. A l ­ though the pCBlOl chimaera (pRBl) appeared to replicate autonomously in both rec+ and rec~ strains of B. subtilis.

it appeared to be present in low copy number. Furthermore,

pRBl, appeared to be unstable as it was rapidly lost from

the population when selection was removed (Collins et a l . , 1985) . Eve n so, at the time of this study pCBlOl still

represented the best candidate for use in the construction of a clostridial cloning vector.

An additional attraction to employing pCBlOl in the intend­ ed vector construction studies was the earlier report that

this plasmid might encode a bacteriocin (butyricin); if so d e t e r m i n e d , it w o u l d r e p r e s e n t the fi r s t i d e n t i f i e d "marked" plasmid from a s a ccharolytic C l o s t r i d i u m . The

suggested role of either of the two co-resident plasmids,

pCBlOl and pCB102, in bacteriocin production (Minton &

Morris, 1981) was p lausible as these two plasmids were isolated from the bacteriocinogenic strain, C. butvricum NCIB 7423 (Clarke & Morris, 1976) , and bacteriocin produc­ tion has been associated with other clostridial plasmids ( G a m i e r & Cole, 1986; Ionesco et a l . . 1974, 1976; Li et al., 1980; Mihlec et a l .. 1978).

For this study it was decided to further characterise the plasmid pCBlOl. By deter m i n i n g the complete nucleotide

sequence of pCBlOl, the o b j e c t i v e s were twofold: (1) to study the region of pCBlOl that appears to comprise the minimal replicon and hopefully gai n some understanding of the mechanisms involved in the replication of this plasmid that might be beneficial to the vector construction pro­

gram, and (2) to examine the remainder of the plasmid for open reading frames ( O RFS), to wh i c h functions might be

open re a d i n g frames ( O R F S ) , to w h i c h functions might be assigned, e.g., bacteriocin production.

3.2.__ RE8PLT8

3.2.1. SUB-CLONING OF THE MINIMAL REPLICON OF pCBlOl

The plasmid isolation techniques described for C. butvricum

(Minton & Morris, 1981) p r o v e d relatively inefficient, resulting in low yields of pCBl O l DNA. The recombinant plasmid pRBl (Collins et a l ., 1985) was therefore chosen as a suitable source of the 3.3 kb S a u 3A fragment of pCBlOl;

propagation of pRBl in E. coli would therefore enable the isolation of a s u f f icient q u a n t i t y of the 3.3 kb S a u 3A fragment for nucleotide sequence determination.

A strain harbouring pRBl (Fig. 3a) was obtained from M.

Young (University of Aberystwyth) and employed for the large s c a l e isol a t i o n of p l a s m i d DNA. The p l a s m i d DNA obtained, however, was found to be substantially smaller than expected, with the largest DNA fragment generated by digestion with Sau3A being only 2.4 kb in size. The deleted

plasmid was designated pRBlAl. A further three independent­ ly isolated clones c a r r y i n g equ i v a l e n t plasmids to pRBl were obtained from M.Young and large scale plasmid prepa­ rations undertaken. On the basis of the sizes of the frag­ ments released after digestion with Sau3A. all three plas­

mids were identical to pRBlAl. These plasmids were there­ fore designated pRBlA2-4.

To determine the extent of the observed deletions extensive

restriction analysis of pRBAl-4 was undertaken. This in­ volved digestion of the four plasmid DNA preparations, both singly and in various combinations, with Hind l l l . T a a l .

Ball, Avail, ACCI. Pstl. H p a l. Ncol , Aatll. and Sau3A. The

FIGURE 3a DERIVATION OF THE CHIMAERIC PLASMID pRBI

The v a r io u s r e g io n s o f th e p lasm ids a r e a s f o l l o w s : --- pBR322 PC 19«

lililí Lambda

'

r

Bel I

cal a n d that the deleted DNA was predominantly of a clos­ tridial origin. The deletion event had resulted in the loss

of a 2.5 kb region of the pRBl restriction map (Fig. 3b) encompassing the clostridial DNA insert.

The results obtained indicated that plasmids generated by inserting pCBlOl DNA into pJABl were structurally unstable in E. c oli. This factor had not come to light in the study

by Collins et a l . (1985), as large scale preparation of

pRBl had not been undertaken (M. Young, personal communica­ tion) . In the study of Luczak et a l . (1985) similar plas­ m ids had been g enerated u s i n g an alternative v e ctor to

pJABl. These resultant plasmids were structurally stable (W.L. S t a u d e n b a u e r , personal communication). This would suggest that it was the cloning vector employed rather than the pCBlOl DNA per se which induced instability on recombi­

nant plasmids. It was therefore elected to sub-clone the 3.3 kb Sau3A fragment of pCBlOl into the smaller and better characterised vector, p U C 8 . Plasmid DNA prepared from C. butvricum NCIB 7423 was digested to completion with Sau3A

and the 3.3 kb DNA fragment derived from pCBlOl isolated

by electroelution. This purified DNA fragment was ligated

with BamHI cut, dephosphorylated, pUC8 DNA and the ligation p r o d u c t s used to t ransform competent E. coli JM83. The p l a s m i d DNA of six p r e s u m p t i v e r e c o m b i n a n t c l o n e s was prepared by the small scale isolation technique and analy­ sed b y digestion with S a u 3A and subsequent agarose gel electrophoresis (1% w / v ) . In each case the presence of a

s i ngle 3.3kb DNA insert was demonstrated. One clone was chosen and employed for the large scale isolation of plas­ mid DNA. Subsequent digestion with S a u 3A and agarose gel electrop h o r e s i s (1% w/v) of this DNA, designated pCBl, indicated that it was structurally stable. Plasmid pCBl is illustrated in Fig. 3c.

FIGURE 3c RESTRICTION ENDONUCLEASE MAP OF dCBI

The v a r io u s r e g io n s o f th e p la s m id s a re a s f o l l o w s : _pUC8 pCB101

3.2.2. NUCLEOTIDE SEQUENCE DETERMINATION OF THE MINIMAL