ELSEVIER FEMS Microbiology Letters 125 (1995) 89-94

Kinetics of cobalamin repression of the cob operon in Salmonella typhimurium

Dan Andersson Uppsala Uniuersiry, Department of Microbiology, BMC, Box 581, S-75123 Uppsala, Sweden

Received 19 September 1994; revised 1 November 1994; accepted 1 November 1994

Abstract

The cob operon in Salmonella fyphimurium encodes 25 proteins involved in the biosynthesis of cobalamin. Expression of the cob operon is negatively feedback regulated by cobalamin via a translational control mechanism. The concentration of cobalamin required to repress cob expression to half-maximal was determined in vivo and in vitro to 0.4 PM and 0.6 PM, respectively. These results suggest that cob expression in wild-type cells is partially repressed by de novo synthesized cobalamin.

Keywords: Cobalamin; cob Operon; Feedback repression; Salmonella typhimurium

1. Introduction

Cobalamin (vitamin B12) is only synthesized de novo under anaerobic growth conditions in

Salmonella typhimurium [ll. Most of the approxi- mately 30 enzymes required to synthesize cobalamin are encoded within the cob operon, located at 41 min on the chromosome [2-41. Expression of the cob operon is regulated by at least four different

effecters: redox [5-71, CAMP 121, propanediol[8-101 and cobalamin [2]. Cobalamin represses cob expres- sion: a plasmid-borne translational cob:: 1acZ fusion

is repressed 30-60-fold and a transcriptional fusion 5-lo-fold. Repression is mediated by a translational control mechanism which requires sequence ele- ments within the 462-nucleotide leader region of the cob mRNA [ll-131. In particular, the 255-380 re- gion is important for correct control and this region was proposed to form an RNA operator site for cobalamin binding [12]. Furthermore, repression re- quires formation of adenosyl-cobalamin and cannot

be mediated by cyano-, methyl- or aqua-cobalamin [14,15].

An understanding of the quantitative properties of the negative feedback loop of cobalamin repression requires knowledge of the rate of synthesis and utilization of cobalamin, as well as the kinetics of the repressing control mechanism. These parameters will determine the steady-state level of cobalamin, the stability of the regulatory loop, and how fast a new steady-state is established when the supply of or demand for cobalamin is altered (ref. [16] and refer- ences therein). One key parameter, which is reported here, is the concentration of cobalamin required to repress cob expression to half-maximal.

2. Materials and methods

2.1. Bacteria, plasmids and genetic techniques

The genotypes of the strains and plasmids used are given in Table 1. All bacterial strains used are

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90 D. Anderson / FEMS Microbiology Letters 125 (1995) 89-94

Table 1

Strains and plasmids used

Strain/plasmid

DA3963

DA3964

DA3965

DA3966

pRS552

pDA3355

Description

metE205, ara-9, reb-1845::TnlO, cobR3/pDA3355

metE205, ara-9, zeb-1845::TnlO. cobR4/pDA335.5

metE205, ara-9, zeb-1845::TnlO/pDA3355

metE205, ara-9, cob-16::TnlO/pDA3355

Translation fusion vector, kanR (EcoRI-BarnHI), lacZ, ?

- 160 to + 776 fragment from Cob promoter region

inserted into the EcoRI-BamHI sites of pRS552

Origin

This work

This work

This work

This work

[231

Ull

pDA3480

pDA3491

Translation fusion vector; ApR, ori, Ptac (EcoRI-BarnHI), lacZ, f

+ 119 to + 687 fragment from Cob promoter region

inserted into the EcoRI-BamHI sites of pDA3480

[121 This work

The isolation and characterization of the cobR3 and R4 mutants is described in [5].

derivatives of Salmonella typhimurium LT2. Genetic [5] and recombinant DNA techniques [17] used in constructing strains and plasmids were as described.

2.2. Growth conditions and assays

Cells were grown anaerobically in glucose mini- ma1 medium with required supplements as described previously [5] when used for /3-galactosidase or cobalamin assays. The various strains showed identi-

cal growth rates and were harvested at log phase,

allowing us to consider the concentrations of p- galactosidase and cobalamin determined as directly proportional to their rate of synthesis. /3-Galactosi- dase and cobalamin were assayed as described previ- ously [5]. Briefly, for cobalamin assays cells were harvested, washed and cobalamin was extracted with HCl. Samples were boiled in the presence of cyanide to convert all cobalamin to cyano-cobalamin. The amount of cobalamin was then determined by com-

40

cob ::Tn 10

30

cobR4

0.2 0.4 006

Intracellular cobalamin concentration (uM)

0.6

Fig. 1. Expression of a translational cob::lacZ fusion in viva as a function of intracellular cobalamin concentration. Each data point

represents the average of at least three separate measurements of the cellular cobalamin concentration and fi-galactosidase activity.

Variation between the measurements was less than f 25% of the average.

D. Andersson / FEMS Microbiology Letters 125 (1995) 89-94 91

peting a known amount of radioactively labelled cobalamin with the sample for binding to porcine intrinsic factor immobilized on agarose beads. To determine the intracellular concentration, cobalamin levels were measured for a volume of culture of known cell titer and the cell volume was calculated as described [18]. To determine repression in vitro, a commercially available coupled transcription-trans- lation system was used according to the manufac- turer’s instructions (Promega). The /3-galactosidase activity was measured after a 20-min incubation (the rate of synthesis of P-galactosidase was linear for at least 40 min).

3. Results and discussion

To determine the concentration of cobalamin re- quired to repress expression to half-maximal in vivo, expression of the cob operon was measured in a set of strains which contain different amounts of cobal- amin (DA3963-3966, see Table 1). The amount of fi-galactosidase produced from a plasmid-borne cob:: lad translational fusion present in these strains

14

12

was used as a measure of repression. The cob pro- moter fragment inserted upstream of the 1acZ gene extends from nucleotides - 160 to + 776, numbered relative to the transcriptional start site, and contains all the regulatory elements required for normal re- pression control [12,13]. As shown previously, regu- lation is not affected by the copy number of the cob operon allowing the use of a plasmid-borne cob:: 1acZ fusion to probe regulation [12,13]. Cells were grown anaerobically in glucose, methionine, ampicillin, M9 minimal medium and Pgalactosidase concentrations were assayed [5]. From the same culture total cobal- amin concentrations were measured by using a com- mercial radioisotope dilution assay as described [5]. This assay measures total cobalamin but as men- tioned previously adenosyl-cobalamin is the repress- ing species. However, adenosyl-cobalamin consti- tutes the major part of the total cobalamin in the cell [151 and the total levels measured here were there- fore approximated to represent mainly adenosyl- cobalamin. In Fig. 1 the cellular concentration of P-galactosidase is plotted as a function of the intra- cellular cobalamin concentration. The cob: :Tn 10 mutant produced the highest amount of /3-galactosi-

0 1 2 3

Concentration of adcnoeyl-cobalrmin (uM)

Fig. 2. Expression of a translational cob::lacZ fusion, in a coupled in vitro transcription-translation system, as a function of

adenosyl-cobalamin concentration. Each data point represents the average of at least three separate measurements. Variation between the

measurements was less than f 25% of the average.

92 D. Andersson / FEMS Microbiology Letters 125 (1995) 89-94

dase which was set as the non-repressed level of expression. The wild-type strain and the cobR3 and

R4 promoter up mutants produced increasing amounts of cobalamin, and showed a corresponding

decrease in cob::lacZ expression. From Fig. 1 the concentration of cobalamin required to repress cob expression to half-maximal was determined to be 0.4 mM.

To determine repression in vitro, a commercially available coupled transcription-translation system was used. This assay system is a modification of the cell-free Escherichia coli S30 extract system de-

scribed by Zubay and co-workers [19]. A plasmid carrying a pt,,.. -.cob:: lad translational fusion

(pDA3491, see Table 1) was used as DNA template. Transcription is driven by the tat promoter in this construct. The cob fragment extends from + 119 to +687 and includes the sequences required for nor- mal repression. In addition, the la& gene is fused downstream of the cob DNA to allow quantitation of repression. Varying amounts of adenosyl-cobalamin was added to the transcription-translation system and the amount of P-galactosidase synthesized was determined. In Fig. 2, /3-galactosidase activity is plotted as a function of the adenosyl-cobalamin con- centration. The level of cobalamin required to re- press cob expression to half-maximal was deter-

mined to be 0.6 PM from the plot shown in Fig. 2. It is not clear why the concentration required to

repress cob expression to half-maximal differ in vivo and in vitro but, given the differences between the two assay systems the discrepancy in values is sur- prisingly small. These values are of interest in at least two contexts. Firstly, they are relevant to how cob expression responds to altered cellular cobal- amin concentrations. The concentration of de novo produced cobalamin in wild-type cells is approxi- mately 0.2 PM (see Fig. 1) and the concentrations of cobalamin resulting in half-maximal expression were determined to be 0.4 mM (in vivo) and 0.6 PM (in vitro). From Figs. 1 and 2 we can extrapolate that, at a cobalamin concentration of 0.2 PM, cob expres- sion in the wild-type cell is approximately 70% of the non-repressed expression. These results would therefore suggest that expression from the cob operon is 30% repressed by de novo synthesized cobalamin. A conceivable regulatory rationale for the partial repression is that changes in the cobalamin level due

to altered supply or demand will directly influence

cob expression. Thus, if exogenous cobalamin is provided, cob expression is decreased concomitantly and costly de novo biosynthesis is shut-down, In

contrast, if the cobalamin concentration resulting in half-maximal expression was considerably higher than the intracellular cobalamin concentration, cob expression would be unregulated over a certain con- centration range and not respond directly to added exogenous cobalamin. Secondly, these values are of

interest in relation to cobalamin transport. The btuB gene encodes an outer membrane protein involved in transport of exogenous cobalamin and the btuB gene is, similar to the cob operon, repressed by cobalamin

[20,21]. Aufrere et al. [22] determined the apparent K, for cobalamin repression of the btuB gene in E. coii to 3 PM. The 5-8-fold higher concentration of cobalamin required to repress expression to half- maximal for the btuB gene compared to the cob operon is presumably physiologically significant. Thus, in order to transport external cobalamin and accumulate sufficient intracellular cobalamin to com- pletely turn off the cob operon and de novo biosyn- thesis the btuB gene has to be repressed at a higher cobalamin concentration than the cob operon. Also, it follows from these values that both the cob operon and the btuB gene are maximally expressed at low cobalamin levels ( < 0.4 PM), serving to increase the

supply of intracellular cobalamin. As the cobalamin levels increase (> 0.4 PM to < 3 PM) cob expres- sion is repressed and costly de novo biosynthesis is

prevented. Finally, at high cobalamin levels (> 3 PM) btuB expression is also repressed, presumably to prevent a build-up of a non-usable excess of cobalamin. In conclusion, the regulation of the cob operon and the btuB gene is tuned so as to provide the cell with sufficient intracellular cobalamin at

varying external cobalamin levels.

Acknowledgements

This work was supported by the Swedish Natural Science Research Council and the Knut and Alice Wallenberg Foundation. I thank Diarmaid Hughes, Kurt Nordstrom and Gerhart Wagner for critical reading and comments.

D. Andersson / FEMS Microbiology Letters 125 (1995) 89-94 93

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