WO2004113373A1 - Overexpression of the cyddc transporter - Google Patents

Abstract

We describe a cell culture system for the enhanced production of amino acids, peptides and polypeptides by bacterial cells and including genetically modified cells for use in said system.

Description

OVEREXPRESSION OF THE CYDDC TRANSPORTER

The mvention relates to a cell culture system for the enhanced production of amino acids, peptides and: polypeptides by bacterial cells and including genetically modified cells for use in said system.

Bacterial expression systems for the production of molecules, in particular amino acids, peptides and polypeptides, are well known in the art. Typically, bacterial host cells are transformed with a vector (e.g. plasmid, phagemid) that is provided with expression signals (e.g. promoter sequences) that are operably linked to a nucleic acid molecule that encodes a polypeptide sequence the expression of which is desired. Vectors are also provided with replication origins that facilitate the replication of the vector inside the host bacterium.

The large scale production of recombinant proteins requires a high standard of quality control since many of these proteins are used as pharmaceuticals, for example: growth hormone; leptin; erythropoietin; prolactin; TNF, interleukins (IL), IL-2, IL-3, IL-4, IL-5, IL-6, ΓL-7, IL-9, IL-IO, ΓL-11; the p35 subunit of IL-12, IL-13, IL-15; granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF); cardiotrophin-1 (CT-1); leukemia inhibitory factor (LIF); oncostatin M (OSM); interferon, IFNα, TFNγ, and extracellular receptor domains from any cell surface receptor. Moreover, the development of vaccines, particularly subunit vaccines, (vaccines based on a defined antigen, for example gpl20 of HIV), requires the production of large amounts of pure protein free from contaminating antigens which may provoke anaphylaxis.

Bacterial expression systems are also used in the industrial production of amino acids, for example the sulphur containing amino acids cysteine and cystine and the tri-peptide anti-oxidant glutathione. The use of bacterial expression systems is known for the industrial production of amino acids. For example, WO0127307 describes the deregulation of the cysB gene resulting in elevated levels of cysteine and cysteine derivatives as a consequence of over- expression of CysB.

US6, 027, 888 describes the bacterial production of di-sulphide containing polypeptides by co-expression of a eukaryotic foldase (e.g. protein disulphide isomerase) to facilitate recombinant protein production.

JPl 1155571 discloses a bacterial strain genetically engineered to suppress the activity of a cysteine desulphydrase, which reduces cysteine degradation in the cell.

The fermentative preparation of L-cysteine, L-cystine, N-acetylserine and thiazolidine derivatives is described in US5, 972, 663. The patent describes bacterial cells transformed with genes involved in the efflux of antibiotics from the cell (e.g. mar locus, emr locus, cmr locus, mex genes) and their over-expression. One of the consequences of over-expression is an increase in the efflux ofthe amino acid cysteine.

In addition to the use of bacterial expression systems to enhance the expression of polypeptides- and amino acids, peptides are also expressed on an industrial scale. For example, WO02061106 describes the transformation of bacteria with a gene encoding serine acetyltransferase and its over-expression. These transformed cells show enhanced production of cysteine, cystine and the tri-peptide glutathione (L glutamate: L cysteine: glycine).

Glutathione (GSH) is an anti-oxidant and is used extensively in the food industry. It is thought to. have beneficial properties with respect to boosting the immune system by neutralising oxygen free radical activity in the body. GSH is usually depleted by oxidative stress which sometimes occurs during trauma, illness or infection. GSH is synthesised in two steps by the enzymes γ-glutamylcysteine synthetase (encoded by gshA) and glutathione synthetase (gshB), and serves as a reductant in many cellular reactions. An important function of GSH is to reduce disulphide bonds, which form within cytoplasmic proteins when exposed to oxidative stress. This phenomenon occurs as a result of exposure to elevated levels of reactive oxygen species such as superoxide (O₂ ^"), hydrogen peroxide (H₂O ), and alkyl hydroperoxides (ROOH) such as cumene hydroperoxides and t-butyl hydroperoxide.

The effects of oxidative damage are severe and can result in DNA damage, lipid peroxidation, disassembly of iron-sulfur clusters, and unwanted disulphide bond formation within proteins. A GSH pool size of approximately 10 mM exists in the cytosol of E. coli at all times and the high ratio of reduced to oxidised glutathione (GSSG) is ensured by the activity of glutathione reductase (gor). There have been several unsupported claims that GSH can, or does not, leak out or be secreted into the periplasmic space, and there are also conflicting views on whether exogenous GSH can be taken up by Gram negative bacteria. Nevertheless, it is not generally assumed that GSH has a physiological role in the oxidising environment ofthe periplasm.

We describe and characterise a mutant bacterial strain that is mutated in genes encoding membrane bound oxidase polypeptides.

Escherichia coli possesses two major membrane-bound terminal respiratory oxidases, namely cytochromes bo ' ("δo₃" encoded by cyoABCDE) and bd, comprising two polypeptide subunits (encoded by cydA and cydB) and hemes bsss, bsps, and d (1-3). Both oxidases catalyse ubiquinol oxidation and oxygen reduction but differ in the efficiency with which electron transfer is coupled to proton translocation (2, 4), and the pattern of expression in response to environment (1, 2, 4). Significantly, cytochrome bd is required for resistance to a number of environmental stresses and its loss attenuates virulence in several bacteria (5, 6). Assembly of cytochrome bd is dependent not only on the structural genes cydAB, but also on the unlinked cydDC operon (7-9). The latter genes are predicted to encode a heterodimeric ABC¹ -type transporter (traffic ATPase) (9) with a previously unknown export function (10, 11). Therefore, unlike traffic ATPases involved in uptake, CydDC is not thought to interact with a cognate periplasmic-binding protein. Strains defective in either cydD or cydC display diverse phenotypes; in addition to loss of cytochrome bd, including loss of periplasmic b- and c-type cytochromes (10, 12), increased sensitivity to H₂O₂ and Zn²⁺ ions (12, 13), and decreased survival in stationary phase cultures (14). It was hypothesised that the substrate of CydDC might be haem (9, 10) that would be assembled into apo-cytochromes following export to the periplasm. However, the assembly of haem into heterologous apoproteins (Ascaris hemoglobin domain 1) exported to the periplasm of E. coli does not require cydC (12), suggesting that outward transport of haem is not absolutely dependent on CydDC. Furthermore, transport studies using inside-out vesicles derived from wild-type and cydD mutant strains revealed no discernible differences between the two strains in association of radio- labelled heme with vesicle membranes (15).

We present evidence that CydDC exports thiol-containing compounds, for example and not by way of limitation, cysteine and glutathione, to the periplasm. We also report further phenotypes associated with a cydD lesion, including loss of motility and increased sensitivity to benzylpenicillin, both of which can be corrected by addition of exogenous L-cysteine. In further support of the cysteine-transport hypothesis, we show that cydD mutant cells have higher cytoplasmic levels of cysteine and are more susceptible to growth inhibition by external cysteine, whereas strains that over-express CydDC exhibit increased resistance to cytotoxic levels of cysteine.

GSH, like L-cysteine, can restore defects associated with periplasmic stress in a cydD mutant. Moreover, a gshA mutant also displays phenotypes indicative of periplasmic redox stress. Assembly of cytochrome d was not restored in a cydD mutant by the exogenous addition of GSH. We demonstrate ATP-dependent uptake of GSH into everted membrane vesicles of a wild type strain and show that such transport does not take place in everted membrane vesicles derived from a cydD strain.

According to an aspect of the invention there is provided a bacterial cell wherein said cell is genetically modified which modification is the transformation of said cell with a nucleic acid molecule wherein said nucleic acid molecule encodes a polypeptide with the specific enzyme activity associated with the CydDC transporter and further wherein said enzyme activity is overexpressed when compared to a non-transformed reference cell ofthe same species.

In a preferred embodiment of the invention said nucleic acid molecule is selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented by Figure 13a and 13b; ii) a nucleic acid molecule which hybridises to the nucleic acid molecule in

(i) and which has the enzyme activity associated with the CydDC transporter; iii) a nucleic acid molecule consisting of a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii) above.

In a flrrther preferred embodiment ofthe invention said nucleic acid molecule hybridises under stringent hybridisation conditions to the sequence presented in Figure 13a and 13b.

Stringent hybridisatiori/washing conditions are well known in the art. For example, nucleic acid hybrids are stable after washing in O.lx SSC, 0.1% SDS at 60°C. It is well known in the art that optimal hybridisation conditions can be calculated if the sequence of the nucleic acid is known. For example, hybridisation conditions can be determined by the GC content of the nucleic acid subject to hybridisation. . A common formula for calculating the stringency conditions required to achieve hybridisation between nucleic acid molecules of a specified homology is:

81.5° C + 16.6 Log [Na⁺] + 0.41[ % G + C] -0.63 (% formamide).

Typically, hybridisation conditions uses 4 - 6 x SSPE (20x SSPE contains 175.3g NaCl, 88.2g NaH₂PO₄ H₂O and 7.4g EDTA dissolved to 1 litre and the pH adjusted to 7.4); 5- lOx Denhardt's solution (50x Denhardt's solution contains 5g Ficoll (type 400, Pharmacia), 5g polyvinylpyrrolidone and 5g bovine serum albumen; lOOμg-l.Omg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecyl sulphate; optionally 40-60% deionised formamide. Hybridisation temperature will vary depending on the GC content ofthe nucleic acid target sequence but will typically be between 42°- 65° C.

In a preferred embodiment of the invention said nucleic acid molecule consists of the nucleic acid sequence presented in Figure 13a and 13b.

In a further preferred embodiment ofthe invention said cell over-expresses said enzyme activity by at least two-fold when compared to a non-transformed reference cell of the same species. Preferably said enzyme activity is over-expressed at least 3-fold; 4-fold; 5-fold; 6-fold; 7-fold; 8-fold; 9-fold; or at least 10-fold. More preferably said enzyme activity is over-expressed at least 20-fold; 30-fold; 40-fold; or at least 50-fold. Preferably said enzyme activity is over-expressed by at least 100-fold.

The over-expression of enzyme activity can be achieved by means known to those skilled in the art. For example, placing the gene encoding an enzyme with the activity ofthe CydDC transporter on a high copy number plasmid. Alternatively, or in addition, said gene can be operably linked to a promoter sequence which provides for high level expression of said gene, said promoter can be constitutively active or inducible. Adaptations also include the provision of selectable markers, which select for cells containing high copy plasmids. These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general.

In an alternative preferred embodiment of the invention said enzyme over-expression is provided by a variant gene which has the activity ofthe CydDC transporter wherein said activity is enhanced when compared to an unmodified reference gene as represented by the amino acid sequence in Figure 14a or 14b.

In a preferred embodiment of the invention said bacterium is transformed with a gene which encodes a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue and wherein said variant polypeptide has the activity associated with the CydDC transporter; preferably said activity is enhanced.

A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.

A functionally equivalent polypeptide is a variant wherein one in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues, or one in which one or more amino acid residues includes a substituent group. Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Tfrr; exchange of the acidic residues Asp and Glu; substitution between amide residues Asn and Gin; exchange ofthe basic residues Lys and Arg; and replacements among aromatic residues Phe and Tyr. In addition, the invention features polypeptide sequences having at least 75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein.

In a further preferred embodiment of the invention said bacterial cell is a Gram negative bacterial cell, for example Escherichia coli.

In an alternative preferred embodiment of the invention said bacterial cell is a Gram positive bacterial cell, for example, a bacterium of the genus Bacillus spp. (e.g. B. subtilis; B. licheniformis; B. amyloliquefaciens).

Gram positive and Gram negative bacteria differ in many respects from one another. A difference exists in the nature of their respective cell walls. The biochemical composition of the B. subtilis cell wall is quite different from that of E. coli. The cell walls of E. coli and B. subtilis contain a framework that is composed of peptidoglycan, a complex of polysaccharide chains covalently cross-linked by peptide chains. This forms a semi-rigid structure that confers physical protection to the cell since the bacteria have a high internal .osmotic pressure and can be exposed to variations in external osmolarity. In Gram-positive bacteria, such as the members ofthe genus Bacillus, the peptidoglycan framework may represent as little as 50% ofthe cell wall complex and these bacteria are characterised by having a cell wall that is rich in accessory polymers such as teichoic acids.

It is known that Gram negative bacteria do not readily secrete polypeptides into the surrounding growth medium although Gram positive bacteria, in particular those of the genus Bacillus spp, do have cell transport mechanisms to secrete polypeptides, these secreted polypeptides can be endogenous polypeptides, (e.g. amylases) or recombinant heterologous polypeptides. However, both are known to have transport mechanisms that transport amino acids and small peptides into the surrounding growth medium. Methods to transform bacteria are well known in the art and have been> established for many years. These include chemical methods (e.g. calcium permeabilisation) or physical permeabilisation (e.g. electro oration).

According to a further aspect of the invention there is provided a cell culture vessel comprising a cell according to the invention and medium sufficient to support the growth of said cell.

In a preferred embodiment ofthe invention said vessel is a fermentor.

According to a further aspect of the invention there is provided a method for the manufacture of at least one molecule comprising the steps: i) providing a vessel comprising a cell according to the invention; ii) providing cell culture conditions which facilitate the growth of a cell culture contained in said vessel; and optionally iii) isolating said molecule from said cell or said cells surrounding growth medium.

In a preferred embodiment of the invention said molecule is a protein. Preferably a protein that contains a sulphur-containing amino acid.

Recombinant protein production relates to the synthesis of protein in expression systems. Heterologous polypeptides include commercially important polypeptides, for example enzymes used in biocatalysis (e.g. restriction enzymes, enzymes used in industrial processing; e.g. amylases, proteases, nucleases, lipases) and therapeutic polypeptides. The large scale production of recombinant proteins requires a high standard of quality control since many of these proteins are used as pharmaceuticals, for . example, interleukins, growth hormone, erythropoietin, and interferon. Moreover, the development of vaccines, particularly subunit vaccines, (vaccines based on a defined antigen,^" for example gpl20 of HIV), requires the production of large amounts of pure protein free from contaminating antigens which may provoke anaphylaxis. The ability to secrete polypeptides into growth medium offers an opportunity- to purify polypeptides without the need for extraction from a host cell expressing said polypeptide.

In a further preferred embodiment of the invention said polypeptide is a peptide, preferably a peptide that includes a sulphur-containing amino acid. Preferably said peptide is glutathione, or a structural variant of glutathione.

In a further preferred embodiment of the invention said molecule is an amino acid, preferably a sulphur-containing amino acid. Preferably said amino acid is cysteine or cystine.

In a preferred method ofthe invention said cell is a Gram positive bacterium.

In an alternative method bf the invention said cell is a Gram negative bacterium.

An embodiment of the invention will now be described by example only and with reference to the following Figures:

Figure 1 illustrates a two-dimensional polyacrylamide gel electrophoresis of polypeptides from periplasmic fractions of wild-type strain AN2342 (A) and cydD mutant AN2343 (B). Arrows identify polypeptides whose levels were found either to decrease (1, 2, 3, 4, 7, 8, 9, 10 in panel A) or to increase (5 and 6 in panel B) in the cydD mutant;

Figure 2 illustrates that exogenous cysteine partially restores swarming behaviour of E. coli cells. Strains AN2342 (cydD⁺) (white bars) and AN2343 (cydD) (black bars) were grown in liquid broth to stationary phase at 30 °C, then inoculated with sterile toothpicks onto LB (0.3% Difco agar) with or without cysteine and incubated at 30 °C for 8 h. Bars show mean values and standard deviations in three experiments; Figure 3 illustrates that exogenous cysteine confers resistance to benzylpenicillin differentially in wild-type and cydD mutant strains. Strains AN2342 (cydD*) (white bars) and AN2343 (cydD) (black bars) were grown at 37 °C to stationary phase. Portions (5 μl) of serially diluted cultures were drop-plated onto LB containing benzylpenicillin (20 μg ml^"1) and/or cysteine (2 mM). Bars show the mean of 10 replicates with standard deviations. Total colony counts are shown; note that in the presence of both penicillin and cysteine, the cydD mutant produced full-sized colonies in 16-18 h whilst the wild- type strain produced tiny 'pin-prick' colonies;

Figure 4 illustrates that exogenous cysteine modulates cytochrome c levels. Strains AN2342 (cydD⁺) (white bars) and AN2343 (cydD) (black bars) were grown anaerobically in the presence of 0, 0.2 and 2 mM cysteine. Periplasmic fractions were isolated and reduced minus oxidised spectra were recorded. Total amounts of c-type cytochromes were calculated; values shown are means of three determinations with standard deviations;

Figure 5 illustrates that mutants, affected in cydD and orf299 are each hypersensitive to exogenous cysteine. Effects of cysteine on the growth of strains AN2342 (cydD⁺, orf299⁺) (A), AN2343 (cydD, orf299⁺) (B), RKP4611 (cydD⁺, orf299) (C) and R P4612 (cydD, orf299) (D). Cells were grown in LB broth at 37 °C in the presence of 0 mM (gray rhombus), 2 mM (black squares), and 5 mM (open triangles) cysteine. The experiment is typical of three sets of growth curves;

Figure 6 illustrates that CydDC is an ATP-driven cysteine transporter in everted membrane vesicles. The cells used were strain R P4611 (cydD⁺, or/299; white circles in A, B, and C) and RKP4612 (cydD, orf299; black circles in A, B, and C). Panels A and B both show ATP-driven uptake for the wild-type strain only in the absence of inhibitors (A) or in the presence of 2 μM CCCP (B). Panel C shows data for strain RKP4611 (white circles, no vanadate; white triangles, + vanadate) and RKP4612 (black circles) in the presence of 50 mM sodium orthovanadate. In each case, the arrow represents the time point at which 10 mM ATP was added. In A, the bars show standard deviations of three experiments. In B and C, a result typical of three replicates is illustrated;

Figure 7 illustrates that strains AN2342 (wild type, black bars) and AN2343 (cydD, white bars) were grown to stationary phase in MOPS minimal media (pH 7.0) supplemented with 40 mM glucose at 30 °C. A 5 μl aliquot of each strain was plated onto LB (0.3% Difco agar) and incubated at 30 °C for up to 2 d. The diameter of the swarm was measured, and the average of three experiments was taken;

Figure 8 illustrates that strains AN2342 (wild type, black bars) and AN2343 (cydD, white bars) were grown to stationary phase in LB at 37 °C. The OD₆₀₀ of each strain was measured and equalised. Serial dilutions (1 x 10^" ) were made of each strain and 5 μl aliquots of each of the diluted cultures were drop-plated onto LB containing benzylpenicillin (20 μg ml^"1) and/or GSH (2 mM). Bars show the mean of 10 replicates with standard deviations. Total colony counts are shown; note that in the presence of both penicillin and GSH, AN2343 (cydD) produced colonies of normal size in 16-18 h whilst the AN2342 (wild-type) produced 'pin-prick' colonies;

Figure 9 illustrates that strains Fragl (wild type) and MJF355 (gshA) were grown to stationary phase in MOPS minimal media (pH 7.0) supplemented with 40 mM glucose at 30 °C. A 5 μl aliquot of each strain was plated onto LB (0.3% Difco agar) and incubated at 30 °C for up to 2 d. The data represent the average of three individual experiments;

Figure 10 illustrates that strains Fragl (wild type, black bars) and MJF355 (gshA, white bars) were grown to stationary phase in LB at 37 °C. The OD₆₀₀ of each strain was measured and equalised. Serial dilutions (1 x 10^" ) were made of each strain and 5 μl aliquots of each of the diluted cultures were drop-plated onto LB containing benzylpenicillin (20 μg ml^"1) and/or GSH (2 mM). Bars show the mean of 10 replicates with standard deviations. Total colony counts are shown; note that in the presence of both penicillin and GSH, MJF355 (gshA) produced colonies of normal size in 16-18 h whilst Fragl (wild-type) produced 'pin-prick' colonies;

Figure 11 illustrates that the everted membrane vesicles used were from strain AN2342 (wild type, black circles in A, B, and C) and AN2343 (cydD, black circles in A, B, and C). Panels A and B both show ATP-driven uptake for the wild-type strain only in the absence of inhibitors (A) or in the presence of 2 μM CCCP (B). Panel C shows data for strain AN2342 (black circles, no vanadate; white triangles, + vanadate) and AN2343 (white circles) in the presence of 50 mM sodium orthovanadate. In each case, the arrow represents the time point at which 10 mM ATP was added; and

Figure 12 illustrates that transport of GSH (black circles) and GSSG (white circles) were each measured in everted membrane vesicles derived from strain AN2342 (wild type). GSSG was obtained by extraction of DTT from [³⁵S]GSH followed by oxidation in air as described in Materials and Methods. Equal concentrations of both [³⁵S]GSH/GSSG and non-radio-labelled GSG/GSSG were used in each assay. The arrow represents the addition of 10 mM ATP to initiate transport;

Figure 13a is the nucleotide sequence of cydD; Figure 13b is the nucleotide sequence of cydC; and

Figure 14a is the amino acid sequence of cydD; Figure 14b is the amino acid sequence of cyd C.

Materials and Methods

Bacterial strains and plasmids

E. coli strain AN2343 carrying the mutant cydDl allele and its isogenic wild-type parent strain AN2342 have been described before (8). Strains RKP4611 and RKP4612 were constructed by PI transduction (16) of the orβ99:±a.n allele from strain MC4100Δ299 (24) into strains AN2342 and AN2343, respectively. Strains RKP2634 and RKP2005 were obtained by transformation of the wild-type and cydD mutant strain, respectively, with plasmid pRP33 (9) that has the cydDC* operon cloned into vector pBR328.

A summary of the strains and plasmids used in this study is provided in Table 1

TABLE 1

Media and culture conditions

Cells were grown in Luria-Bertani (LB) broth (pH 7.0) (18), or in MOPS-buffered minimal medium (pH 7.4) supplemented with 40 mM lactose or glucose (19) plus 10% (v/v) LB. Kanamycin and benzylpenicillin (penicillin G) were added to give final concentrations of 30 μg ml^"1 and 20 μg ml^"1, respectively.

L-Cysteine was added as a filter-sterilised 100 mM stock solution to media, giving the final concentrations in the text. Aerated cultures were grown in Erlenmeyer flasks containing one fifth their volume by shaking (200 rpm) at 30 °C or 37 °C. Aαaerobically-grown cultures were obtained by filling growth vessels to the brim with LB (supplemented with 20 mM KNO₃) and incubating without shaking at 37 °C for 14 h. Where reduced glutathione was added to the media, the concentrations are described in the text. Agar and other dehydrated media were from Difco and Oxoid. Other chemicals were from Sigma. Aerated cultures were grown with shaking (200 rpm) at 30 °C or 37 °C.

Motility assays

Cells were grown to stationary phase in LB broth or MOPS minimal media (pH 7.0) at 30 °C, and 5 μl drops were spotted onto semi-solid LB medium (0.3% Difco agar). The cells were incubated at 30 °C for up to 3 d, and the diameter of the resultant swarm of growth was measured.

Benzylpenicillin sensitivity assays

Cells were grown to stationary phase in LB broth at 37 °C and serial dilutions in 1 ml aliquots were made. Portions (5 μl) of serially diluted suspensions were drop-plated onto solid LB medium containing benzylpenicillin (20 μg ml^"1) and 0.5, 1, 1.5 or 2 mM cysteine. Plates were incubated overnight at 37 °C and the colonies were counted.

Preparation of sub-cellular fractions

Periplasmic fractions were isolated using a modified procedure of Willis et al (20). In brief, 200 ml culture was conditioned for osmotic shock by the addition of 6 ml 1 M NaCl and 6 ml 1 M Tris-HCl buffer (pH 7.3). An equal volume of a 40% (w/v) sucrose solution containing 33 mM Tris-HCl (pH 7.3) and 2 mM EDTA was added, and incubated at room temperature for 20 min. Cells were harvested and to each pellet 6 ml ice cold water was added. After 45 s on ice, MgCl₂ was added to 1 mM and the cells kept on ice for 10 min. Finally, the periplasmic fraction was obtained by centrifugation (10,000 x g for 5 min) at 4 °C to remove cell debris and stored at 4 °C until ready for use. The cytoplasmic fraction for enzyme assays was produced from the pellet (spheroplasts), which was resuspended in a buffer (6 ml) that contained (final concentration) 20 % (w/v) sucrose, 200 mM Tris-HCl (pH 7.5), and 1 mM Na EDTA. Sonication (15 μm amplitude, 4 to 5 x 15 s bursts, with 30 s breaks) on ice was followed by centrifugation (100,000 x g for 70 min) and the resulting supernatant (cytoplasm) was stored at 4 °C. For assay of cysteine in the cytoplasm, 400 ml culture was used and the spheroplasts were suspended in 1 ml water. Centrifugation after sonication was at 200,000 x g for 2 h.

Marker enzyme assays

Assays of β-galactosidase and alkaline phosphatase activities were used to determine the purity of periplasmic and cytoplasmic fractions, β-galactosidase (18) and alkaline phosphatase (21-22) activities were measured at room temperature by monitoring at 420 nm the hydrolysis of o-nitrophenyl-β-D-galactopyranoside or 4-nitrophenyl phosphate, respectively.

Two-dimensional gel electrophoresis

Periplasmic samples were concentrated approximately 2-fold with a Centricon YM-3 centrifugal filter device (Amicon Bioseparations - Millipore Corporation) with a maximum volume of 2 ml and a molecular mass cut-off of 3,000 Da. A portion (2 ml) of each sample was spun (5,000 x g for 120 min) without the retentate vial. A further 2 ml of sample was centrifuged exactly as above, and samples were pooled. Concentrated periplasm (approximately 0.2 mg protein) was included in 125 μl (total volume) of rehydration solution (8 M urea, 2 % (w/v) CHAPS, 0.5 % (v/v) JPG buffer pH 3-10 (non-linear) (Amersham Pharmacia Biotech), 0.28 % dithiothreitol, and a few grains of bromophenol blue) and applied to a 7 cm PG strip. After rehydration (18-20 h) 2D gel electrophoresis was carried out using a Multiphor II horizontal unit with immobilised pH gradients (pre-cast JPG strip, pH 3-10, non- linear) in the first dimension and a sodium dodecyl sulfate (SDS)-polyacrylamide gel (8-18 %> polyacrylamide) in the second dimension, according to the manufacturer's instructions (Amersham Pharmacia Biotech). Gels were stained with Coomassie Blue.

Determination of N-terminal sequence

Proteins were electroblotted onto ProBlott (Applied Biosystems) membranes at 400-500 mA for 1.5-2 h before staining with Coomassie Blue. The N-terminal sequences ofthe protein spots were determined by sequential Edman degradation (23). Sequence identity was computed using the Colibri website (http://genohst.pasteiir.fr/colibri7) FASTA function (24). Further information on sequenced proteins was found on the SWISS- PROT website (http Awww.expasy.cbT) .

Cysteine assay

This was carried out using the method of Gaitonde (25). A standard curve (0-0.5 μmol cysteine-HCl) was prepared, and used to quantify cysteine levels in cytoplasmic fractions, which had been treated with acetic acid and acid ninhydrin "Reagent 2" (250 mg ninhydrin dissolved in a mixture of 6 ml acetic acid and 4 ml HC1). Samples were heated in a boiling water bath for 10 min, then cooled rapidly in water before dilution to 5 or 10 ml using 95 % ethanol. After 30 min at room temperature, the reaction products were measured at 561 nm. To correct for interference by other ninhydrin-reactive components that contributed to a sloping baseline in the absorbance spectra of dilute cytoplasmic fractions, A₅₆ι was measured relative to a baseline drawn between 530 and 590 nm.

Cytochrome d and c assays

Cytochrome d was quantified in cells grown aerobically to stationary phase in 50 ml LB and harvested at 6000 x g for 15 min. Cells were washed with 100 mM K-phosphate buffer (pH 7.2) and used to record reduced minus oxidised difference spectra and CO + reduced minus reduced difference spectra at room temperature as before (8) except that a SDB4 dual wavelength scanning spectrophotometer (26) was used. An absorption coefficient ε (622 minus 644 nm) of 12.6 mM^"1 cm^"1 (27) was used. For c-type cytochromes, periplasmic fractions were isolated as described above to minimise interference by other cytochromes with overlapping spectral features. Reduced minus oxidised difference spectra at room temperature were recorded (10) but in the SDB4 dual wavelength scanning spectrophotometer. Correction for baseline drift in the Soret region was accomplished by dropping a vertical from the absorption peak at about 423 nm (NrfA has a maximum in absolute spectra at 420.5 nm; 28) to a baseline drawn between 404 and 450 nm. The absorption coefficient ε used was 146 mM^"1 cm^"1 (10), determined by using the absorption coefficient ε_{5 1-540} for the α-band (10) and a γ/α ratio of 7.5 measured in spectra of concentrated periplasmic fractions. Protein contents of cell suspensions and periplasmic fractions were assayed using the method of Markwell et al (29).

Oxidation of reduced glutathione to obtain GSSG

The procedure of Butler et al (30) was employed to oxidise GSH. In brief, 0.132 nmol [³⁵S]GSH (28.1 TBq mmol^"1; 760 Ci mmol^"1, Amersham-Pharmacia) which contains 10 mM DTT was acidified with HC1 to pH 2.0 and the DTT extracted with a 10-fold volume of ethyl acetate by shaking for 2 min followed by centrifugation for 1 min. The aqueous phase was retained. This was repeated three times and the aqueous phase was assayed for thiols by the method of Anderson (31).

Preparation of everted membrane vesicles

Up to 6 1 of culture was grown aerobically at 37 °C to the mid-exponential phase of growth (OD₆oo = 0.6) in MOPS minimal medium supplemented with lactose and LB. Cells were harvested by centrifugation and the cell pellet washed with pre-cooled 10 mM Tris-HCl (pH 7.5), containing 140 mM choline chloride, 0.5 mM dithiothreitol, and 10% glycerol (v/v) followed by resuspension in the same buffer (5 vol/g of wet cells). Everted vesicles were prepared by the method of Ambudkar et al (32). In brief, cells were disrupted by a single passage through a French pressure cell at 4000 p.s.i. (34.5 MPa). Pancreatic DNase and MgCl₂ were added at final concentrations of 0.1 mg ml^"1 and 2.5 mM, respectively, and the mixture was incubated on ice for 1 h or until the viscosity decreased significantly. After centrifugation at 10,000 x g for 10 min, vesicles were sedimented from the supernatant by centrifugation at 150,000 x g for 1 h. Vesicles were gently washed once in the same buffer, collected by centrifuging and resuspended to 15-20 mg protein ml^"1. Aliquots (100 μl) were diluted with an equal volume of glycerol before snap-freezing and storage at -20 °C. [¹⁴C]Lactose, [³⁵S] cysteine and [³⁵S] lutathione transport assays

[¹⁴C]Lactose (2109 MBq mmol^"1) and [³⁵S]cysteine (3145 MBq mmol^"1' Amersham- Pharmacia) were added to final concentrations of 0.06 mM and 0.5 mM respectively in the transport assay. In addition, non-labelled lactose and cysteine were added at final concentrations of 1.94 mM and 0.5 mM, respectively. [³⁵S]GSH (28.1 TBq mmol^"1; 760 Ci mmol^"1, Amersham-Pharmacia) was added to achieve a final concentration of 10 nM in the transport assay and, in addition, non-labelled glutathione was added at a final concentration of 1.0 mM. Everted vesicles were thawed slowly on ice and diluted to 1.0 mg protein ml^"1 in 10 mM Tris-HCl (pH 8.0) containing 140 M choline chloride and 5 mM MgCl₂. Vesicles were added to glass tubes containing buffer (pre-equilibrated at 30, °C) to a final volume of 200 μl, and were incubated at 30 °C for 15 min without shaking. To initiate [¹⁴C]lactose transport, vesicles were energised for 15 min prior to lactose addition with 20 mM D-lactate. [ SJcysteine transport was initiated by the addition of cysteine for 5 min prior to the addition of 10 mM ATP. Vesicles were de- energised with either CCCP (2 μM) to dissipate the proton gradient (15), or sodium orthovanadate (50 μM), an analogue of inorganic phosphate that mimics the γ-phosphate of ATP in the transition state for ATP hydrolysis (33). Transport was terminated by rapidly pouring the contents onto cellulose-nitrate filters (0.45 μm pore size), which were washed twice with 4 ml 100 mM LiCl, and dried. Radioactivity was measured by liquid scintillation counting. To minimise non-specific binding of substrate to filters, the filters were pre-soaked in 100 mM LiCl.

EXAMPLE 1

Periplasmic fractions of wild-type and cydD mutant strains have different levels of periplasmic transport proteins

The periplasm of a cydC mutant is more oxidised than that of a wild-type strain (12). Interestingly, the cydDC operon is adjacent to the trxB (thioredoxin reductase) gene (34) on the E. coli chromosome and Goldman et al. (12) suggested that TrxB might be a substrate for CydDC. However trxB (and trxA) mutants do synthesise cytochromes c and bd (10, 15), ruling out TrxB as a substrate of CydDC.

We therefore sought a protein that might be transported by CydDC using 2D-SDS PAGE and N-terminal sequencing to analyse periplasmic fractions of wild-type and cydD strains (Fig. 1 and Table 1). Marker enzyme assays on both periplasmic and cytoplasmic fractions revealed <5 % contamination by cytoplasmic and periplasmic enzymes, respectively (results not shown). Comparison of 2D gels revealed several major differences and, of the spots chosen for excision and subsequent Edman degradation, all were found to be periplasmic proteins, the determined sequences of which began after a signal sequence. This strongly suggests that all proteins identified were exported from the cytoplasm to the periplasm by a Sec-dependent mechanism (35). The proteins, represented by spots I and 9 were identified as OppA (36) and AnsB (37), respectively, and were expressed at significantly higher levels in the periplasm of the wild-type than that ofthe mutant (Fig. 1). A minor spot (number 8) was also OppA and may result from post-translational alteration or modification of lysine residues during electrophoresis (38). Proteins OsmY (39) and HisJ (40) (spots 5 and 6 respectively) were expressed at slightly more elevated levels in the cydD mutant periplasm compared to that of the wild-type (Fig 1). The remaining five sequenced proteins (MalE, GlnH, ProX, HisJ, and DppA) were expressed at slightly higher levels in the wild-type compared to the cydD mutant periplasm and are the periplasmic binding-proteins of secondary type transport systems in E. coli (41 and references therein). Transport mechanisms for all of these proteins are already established, so it seems unlikely that they are substrates ofthe CydDC transporter.

EXAMPLE 2

A cydD mutant displays a cysteine-reversible defect in motility

Many of the well-documented phenotypes associated with loss of CydDC are actually attributable to the consequent loss of cytochrome bd (42). These include sensitivity to H₂0₂ and Zn²⁺ (13) and inability to grow or exit stationary phase at 42 °C (13, 14, 43). Loss of periplasmic c-type cytochromes, however, appears directly attributable to the loss of the CydDC transporter (10). We report here an additional phenotype of a cydD mutant, namely • a defect in motility. When inoculated onto semi-solid agar and incubated overnight at 30 °C, a temperature that enhances expression of flagellar genes (44), the mutant was non-motile (Fig. 2), spreading only slightly beyond the inoculation site. In comparison, the wild-type strain displayed a normal swarming phenotype, producing a halo approx. 50 mm diameter (Fig. 2), punctuated with concentric rings, a characteristic associated with swarmer cell morphology (45).

Thiol compounds have been shown to correct lesions caused by mutations in dsbA and dsbB (46). These genes ^"encode thio oxidoreductases (47, 48) that, with DsbD, control the redox balance in the periplasm by maintaining the cysteine residues of periplasmic C-X-X-C motifs as disulfides. We therefore postulated that cysteine might reverse the lack of motility. The cydD mutant was inoculated onto LB (0.3% agar) containing 2 mM L-cysteine and incubated at 30 °C for 8 h. This strain was non-motile (see above) but, in the presence of cysteine, a zone of swarming, typically 18 mm (Fig. 2), was observed. In contrast, the ability ofthe wild-type to swarm decreased in the presence of cysteine (Fig. 2).

EXAMPLE 3 Effects of cysteine on penicillin sensitivity

Sensitivity to benzylpenicillin is observed when there are defects in disulfide bond formation in the periplasm (51). Cultures of strains AN2342 (wild-type) and AN2343 (cydD), at similar culture densities, were challenged with benzylpenicillin (20 μg ml^"1) in the presence or absence of 2 mM cysteine. In the absence of both penicillin and cysteine, the number of viable cells was slightly lower for the cydD mutant (corresponding to 1.3 x 10⁹ cells (ml culture)^"1) than for the wild-type parent (2.1 x 10⁹ cells (ml culture)^"1), consistent with the compromised viability of cydDC mutants (14). Extracellular cysteine alone slightly increased the viable counts of both strains. Although both strains were incapable of growth on plates containing penicillin alone, (Fig. 3), cysteine suppressed the antibacterial effect more markedly for the cydD mutant than for the wild-type. Importantly, the colony morphologies of the two strains when plated on cysteine with penicillin were markedly different: cysteine allowed growth of the cydD mutant to give normal colonies (1-2 mm diam) after overnight incubation of the plates, whereas colonies of the wild-type strain were extremely small (<0.5 mm) even after prolonged incubation. The suppressing effect of cysteine on the inhibition by penicillin of growth of the cydD mutant was dose-dependent in the range 0.5 to 2 mM cysteine (not shown).

EXAMPLE 4

Dithiothreitol sensitivity of a cydD mutant

The above data demonstrate that a cydD mutant exhibits defects that are reversed by exogenous cysteine, suggesting that maintenance of an appropriate cysteine concentration or redox poise is essential for normal physiology. Consistent with this view is the finding (42) that a cydC mutant is hypersensitive to DTT, a powerful reductant (mid-point redox potential of approximately -330 mV (50)) that is used for determining sensitivity to disulfide bond formation (49). We confirmed that a cydD mutant is also DTT-sensitive: zones of growth inhibition for wild-type and cydD strains were recorded around sterile filter discs soaked in 7 and 15 mM filter-sterilised DTT. This reductant exerted a dose-dependent inhibition of the wild-type strain but, in the cydD mutant, the diameter of the zone of inhibition was three-fold higher than for the wild-type strain at both concentrations. Thus, in the absence of other environmental stresses, the cydD mutant is DTT-sensitive, as well as cysteine-sensitive (see later). .

EXAMPLE 5

Exogenous cysteine modulates levels of c-type cytochromes

Assembly of c-type cytochromes takes place in the oxidising environment of the periplasm and thus presents the cell with a conundrum, since cysteine residues of the apo-cytochrome must be reduced before ligation of heme to the apo-cytochrome (51- 53). DsbD is an integral membrane protein and translocates electrons from the cytoplasm to the periplasm (54), thereby providing the reducing power to enable apo- cytochromes to ligate heme (55). Loss of DsbD results in a loss of c-type cytochromes

(46) and can be corrected by the addition of thiol-containing compounds (56). Poole et al (10) reported that periplasmic c-type cytochromes were barely detectable in a cydD mutant: the Soret absorbance at 418.5 nm (77 K) was less than 10%> of wild-type levels and the distinctive α-band at 550 nm (77 K, expected at 552 nm at room temperature) was undetectable. Since cysteine corrects defects in motility and modulates sensitivity to benzylpenicillin in a cydD mutant (see above), the effects of cysteine on levels of c-type cytochromes were investigated. Wild-type and cydD strains were grown anaerobically in LB plus 20 mM KNO to elevate cytochrome c levels, without or with cysteine (0.2 and 2 mM) supplements. Reduced minus oxidised difference spectra of the periplasm from the wild-type strain showed a Soret band at 423 nm, a β-band at 525 nm, and a - band at 552.5 nm (data not shown). These signals are attributable to the NrfA/NrfB cytochrome c nitrite reductase, maxima for reduced NrfA being, for example, 420.5,

523.5 and 552 nm (35), whilst NrfB has a sharp absorbance maximum at 551 nm, all at room temperature (D J Richardson, personal communication). Cysteine (0.2 and 2 mM) in the growth media was without effect on the band positions but progressively decreased cytochrome c concentration in the periplasm of the wild-type strain (Fig. 4).

Qualitatively similar' spectra were also recorded for the cydD mutant but, in the absence of cysteine, the cytochrome c level was 30 % of the wild-type level. In previous work

(10), we could find no periplasmic cytochrome c in the cydD mutant; the difference may be attributable to the use of fumarate as an electron acceptor (10) or the poorer sensitivity of the earlier spectrophotometer; In the cydD mutant, cytochrome c levels were increased approximately 1.7-fold when cells were grown with 0.2 mM cysteine compared to without, but decreased at 2 mM cysteine (Fig. 4). The opposing effects of

0.2 mM cysteine on the wild-type (decreased cytochrome c) and the cydD mutant strains

(increased cytochrome c) again suggest the significance of an appropriate cysteine- modulated redox poise in the periplasm. EXAMPLE 6

Exogenous cysteine does not restore cytochrome bd in a cydD mutant

Since cysteine increased cytochrome c levels in a cydD mutant, we hypothesised that cysteine might also restore cytochrome bd. CO difference spectra ofthe wild-type strain showed a band at 644 nm, corresponding to the carbonmonoxy form of cytochrome d, as described before (8, 9). Cytochrome bd levels (approx. 0.05 nmol mg protein^"1) were unaffected by the addition of exogenous cysteine (0.2 and 2 mM). Spectra of the cydD mutant revealed no cytochrome d signal at 644 nm and cells grown in the presence of 0.2 and 2 mM cysteine also lacked cytochrome d.

EXAMPLE 7 cydD and orf299 mutants are sensitive to cysteine

Delaney et al (13) reported that htrD (cydD) mutants show growth defects in the presence of cysteine, and that there is increased cysteine uptake compared with a wild- type strain. We therefore considered the possibility that the cysteine sensitivity of cydDC mutants and the ability of exogenous cysteine to modulate physiology (Figs. 2, 3) and cytochrome assembly (Fig. 5) are linked, and result from the failure of cydDC mutants to export cysteine. The effect of cysteine on the growth of a cydD mutant was therefore compared to the effect on the orf299 mutant recently described by Dassler et al (17). This gene product is a putative member ofthe major facilitator superfamily (MFS) of transport proteins and its expression promotes cysteine and OAS excretion (17). To clarify the role(s) of each gene in cysteine metabolism, isogenic strains were first constructed by transducing the orf299 allele into wild-type and cydD mutant strains. The resulting strain RKP4611 (or/299) was insensitive to EDDHA as anticipated for Cyd⁺ strains (57) and CO difference spectra revealed the presence of cytochrome d. However, RKP4612 (or/299 cydD) was sensitive to EDDHA and cytochrome d was absent. The growth phenotypes of the orf299 and cydD orf299 strains were compared to those of wild-type and cydD mutant strains in the presence of 0, 2, and 5 mM cysteine (Fig. 5). Growth of the wild-type was not substantially affected by cysteine. At 5 mM cysteine, there was a slight lag in reaching the stationary phase but optical density was not significantly different after 8 h (Fig. 5A). Growth of the cydD mutant was slightly inhibited at 2 mM cysteine, as characterised by a slower growth rate and a reduction in optical density after 8 h. However, 5 mM cysteine was severely inhibitory and growth was arrested after 4 h (Fig. 5B). The or/299 mutant displayed an extended lag phase and did not reach mid-exponential growth until 3-4 h after inoculation compared to 2 h for the wild-type and cydD strains. Cysteine (5 mM) extended the lag phase by about 1 h. The double mutant (orβ99 cydD) displayed growth that was highly sensitive to cysteine. At 2 mM, growth was arrested after 4 h, and at 5 mM, OD₆₀₀ was further reduced (Fig. 5D). Therefore a cydD mutant strain displays greater cysteine sensitivity than an orf299 mutant, but defects in both genes result in extreme sensitivity to cysteine. The data suggest that both gene products are involved in cysteine resistance.

EXAMPLE 9

Cysteine is transported by CydDC in an ATP-dependent manner

In view of the ability of cysteine to reverse some of the pleiotropic defects of a cydD mutant, particularly those associated with periplasm physiology, it was considered a candidate substrate of CydDC. To test this, we measured uptake of [³⁵S]cysteine by everted membrane vesicles ofthe orf299 and cydD orβ99 strains. Extensive studies (32, 58-60) have already demonstrated the efficacy of French pressure cell treatment or ultrasonication of Gram-negative bacteria in producing predominantly everted (inside- out) vesicles that actively take up solutes, particularly toxic metal or metalloid ions, that would be exported in vivo. To demonstrate that the everted vesicles support active transport under the present conditions, the ability of the vesicles to accumulate [¹⁴C]lactose in response to an energised membrane was measured. The lactose transporter, LacY, can support lactose transport in either right-sided or everted vesicles, provided a protonmotive force (Δp) of the appropriate polarity is applied. Everted vesicles prepared from cells grown in MOPS medium supplemented with glucose have a low rate of lactose transport (15). • Therefore, everted vesicles were prepared from the orβ99 strain grown with lactose. Significant accumulation of [¹⁴C]lactose occurred only if D-lactate was added as an energy source; typical rates of [¹⁴C]lactose transport were 0.18 nmol min^"1 (mg protein)^"1' (data not shown). The addition of the protonophore, CCCP, abolished transport, demonstrating that accumulation of [¹⁴C]lactose was dependent upon Δp (data not shown).

The role of CydDC in cysteine transport was investigated in an orβ99 genetic background, given the presumed role of orβ99 in exporting cysteine from cells and thus into vesicles. [³⁵S]cysteine uptake assays were done using everted membrane vesicles from the orβ99 and cydD orβ99 strains. Since ATP does not permeate the lipid membrane and is hydrolysed only on the inner aspect of the membrane (60), the ability of ATP to drive transport can be taken as evidence of vesicle inversion. In the 5 min period prior to ATP. addition, no uptake of [³⁵S]cysteine was observed in the orβ99 vesicles (Fig. 6A). Upon addition of ATP, however, [³⁵S]cysteine uptake was rapid with maximal uptake occurring over a 3 min period following ATP addition (Fig. 6A). The maximum rate of [³⁵S]cysteine uptake observed under the conditions tested was 0.31 nmol min^"1 (mg protein)^"1. The uptake of [³⁵S]cysteine reached a maximal level 4 min after the addition of ATP and apparent saturation was observed (Fig. 6A). In contrast, no [³⁵S]cysteine uptake was observed in vesicles of RKP4612 (cydD orβ99) before or after the addition of ATP (Fig. 6A).

That the CydDC transporter derives energy for transport directly from ATP was tested with transport inhibitors. CCCP (2 μM) had no discernible effect on the uptake of [³⁵S] cysteine by vesicles of the orβ99 mutant (Fig. 6B), and the rate of [³⁵S]cysteine uptake was approximately 0.21 nmol (mg protein)^"1. There was no [³⁵S]cysteine uptake into everted vesicles of RKP4612 (cydD or/299) in the presence of CCCP (Fig. 6B). Incubation of orβ99 vesicles with sodium ortho vanadate abolished transport such that the rate was indistinguishable from that observed with everted vesicles of RKP4612 (cydD orβ99) (Fig. 6C). These studies confirm that the CydDC transporter does not derive energy from the Δp but directly from ATP hydrolysis. EXAMPLE 10

Over-expression of CydDC confers resistance to exogenous cysteine toxicity

The finding that, in vivo, CydDC exports cysteine and that a cydD mutant is hypersensitive to exogenous cysteine (Fig. 5) predicts that over-expression of the transporter might confer additional resistance at very high concentrations of extracellular cysteine. We have already demonstrated that expression of the entire cydDC operon under the control of its own promoter on a multicopy plasmid results in increased levels of the cydDC transcript and elevated levels of the CydD protein detectable by a polyclonal antibody (61). We therefore compared the growth of various strains in liquid medium containing a cysteine concentration that barely permitted growth of the wild-type strain. At 20 mM cysteine, poor growth of the wild-type and cydD mutant strains was observed 3h after inoculation (Table 2) but, after 9h, culture turbidity declined markedly. Control cultures in the absence of cysteine showed little difference between these two strains at 3 or 9 h. Strain RKP2634, a wild-type strain harboring plasmid pRP33 (cydDC*), showed slightly better growth than the corresponding plasmid-free sfrain at 3h and continued to grow over the 9 h course ofthe experiment. Similar results were obtained when this plasmid was present in a cydD mutant background (strain RKP2005) showing that the plasmid not only complemented the chromosomal mutation but also conferred enhanced cysteine resistance.

EXAMPLE 11

Mutation of cydD or orβ99 increases cytoplasmic pool sizes of cysteine

A further prediction of the hypothesis that both CydDC and the orβ99 gene product contribute to cysteine export from the cytoplasm is that the cytoplasmic pool size of cysteine should be elevated in these mutants. Periplasmic fractions from various isogenic strains were removed by osmotic shock and a cytoplasmic fraction was prepared by disruption of the resulting spheroplasts and removal of membrane material by high-speed centrifugation. In the wild-type strain, the cysteine concentration of the cytoplasm was 0.38 nmol cysteine (mg protein)^"1. This level was elevated 1.6-fold in the cydD mutant, 1.3-fold in the orβ99 mutant (R P4611) and 2.2-fold in the double mutant (RKP4612). In a replicate experiment, similar elevations of cysteine content relative to the wild-type were observed. In an attempt to prevent cysteine oxidation to cystine during extraction, the use of DTT was explored but this reagent interfered with the colorimetric cysteine assay. Despite potential complications from loss of cysteine during cytoplasm preparation or cysteine pool turnover, these data strongly suggest that both CydDC and the orβ99 gene product contribute to cysteine export to the periplasm.

EXAMPLE 12

Glutathione complements defects associated with redox stress in the periplasm

We have previously reported several unidentified phenotypes of a cydD mutant that are concurrent with periplasmic redox stress. These include sensitivity to benzylpenicillin and poor motility. The addition of the thiol compound L-cysteine to semi-solid media partially restored the swarming phenotype of a cydD mutant. In addition, the survival of a cydD mutant when challenged with benzylpenicillin was dramatically increased in the presence of L-cysteine.

Reduced glutathione was tested to see if it could restore motility and increase the survival of a cydD mutant in the presence of benzylpenicillin. AN2342 (wild type) and AN2343 (cydD) were grown to stationary phase at 30 °C in MOPS minimal media, supplemented with 40 mM glucose as described in Materials and Methods. A 5 μl aliquot of each strain was plated onto semi-solid agar containing one of the following reduced GSH concentrations 0, 1, and 2 mM, and incubated at 30 °C for a period of 2-3 days. At 0 mM GSH, the zone of swarming in AN2342 was approximately 58 mm after 2 days (Fig. 7). The zone of swarming for AN2343 at 0 mM GSH was severely reduced compared to AN2342 and was only ^'5 mm from the site of inoculation (Fig. 7). At 1 mM GSH, the zone of swarming of AN2342 is 50 mm and is therefore slightly reduced compared to growth when no GSH is present. Conversely, the zone of swarming in AN2343 in the presence of 1 mM GSH was approximately 27 mm. At 2 mM GSH, the zone of swarming of AN2342 is further reduced and is approximately 42 mm. Under these growth conditions, the zone of swarming of AN2343 was approximately 34 mm. The ability of AN2342 to swarm on semi-solid agar is inhibited by the concentrations of GSH tested, while that of AN2343 is greatly increased as the concentration of GSH increased.

Hypersensitivity to benzylpenicillin results when there are defects in disulfide bond formation in the penicillin-binding-protein-4 (PBP4). To determine if, like L-cysteine GSH can correct this defect, 5 μl aliquots of 10^"6 serial dilutions of strains AN2342 (wild-type) and AN2343 (cydD) were challenged with benzylpenicillin (20 μg ml^"1) in the presence or absence of 2 mM GSH as described in Materials and Methods. All plates were incubated at 37 °C for 14 h. In the absence of both penicillin and cysteine, the numbers of viable cells obtained on plating out culture samples was slightly lower for the cydD mutant (corresponding to 1.2 x 10⁹ cells (ml culture)^"1) than for the wild- type parent (1.7 x 10⁹ cells (ml culture)^"1) (Fig. 8), and this is consistent with our previous observations when cydD mutants were challenged with benzylpenicillin in the presence of L-cysteine. Interestingly, the addition of GSH alone had a deleterious effect upon the viability of AN2342 (1.0 x 10⁹ (ml culture)^"1) (Fig. 8), but greatly increased the viability of the cydD mutant (1.9 x 10⁹ (ml culture)^"1) (Fig. 8). Both strains showed no growth on plates containing benzylpenicillin alone, as expected (Fig. 8), but when grown in the presence of the drug and GSH together the viability of the cydD mutant was substantially greater than that ofthe wild-type (1.5 x 10⁹ (ml culture)^"1 and 2.5 x 10⁸ (ml culture)^"1 respectively) (Fig. 8). As observed with growth in the presence of L- cysteine and benzylpenicillin, GSH allowed growth of the cydD mutant to give normal colonies (1-2 mm diameter) after 14 h incubation of the plates, whereas colonies of the wild-type strain remained as 'pin-prick' colonies even after prolonged incubation. EXAMPLE 13

A gshA mutant displays phenotypes associated with redox stress

If GSH were the substrate of the CydDC transporter, then a gshA mutant would be expected to display phenotypes identical to that of a cydD mutant. Therefore, Fragl (wild type) and MJF355 (gshA) were grown to stationary phase in MOPS minimal media supplemented with 40 mM glucose as described in Materials and Methods. A 5 μl aliquot of each strain was inoculated onto semi-solid agar as described in Materials and Methods. The strains were incubated at 30 °C for 2 days, and the zones of swarming were measured. Fragl displayed a zone of swarming approximately 45 mm in diameter (Fig. 9), while MJF355 (gshA) produced a swarm diameter of 15 mm (Fig. 9).

The gshA mutant was challenged with benzylpenicillin in the presence of glutathione to determine if the patterns of hyper-resistance in the presence of glutathione observed for cydD are also evident in a gshA mutant. An identical set of conditions as those used to test sensitivity of AN2342 and AN2343 to benzylpenicillin were employed for Fragl (wild type) and MJF355 (gshA) as described in Materials and Methods. The plates were incubated at 37 °C for 14 h. In the absence of benzylpenicillin and glutathione, the gshA mutant showed a decrease in viability compared to that of the isogenic wild type strain (1.1 x 10⁹ and 1.9 x 10⁹ (ml culture)^"1 respectively) (figure 4). In the presence of 2 mM glutathione, the viability ofthe gshA strain increased to almost the level ofthe wild type grown on LB alone (1.7 x 10⁹ (ml culture)^"1 (figure 4). Fragl showed a slight decrease in viability when grown in the presence of 2 mM glutathione (1.75 x 10⁹ (ml culture)^"1) (figure 4). There was no growth of either strain in the presence of benzylpenicillin (20 μg ml^"1) (figure 4), as expected. However when challenged with benzylpenicillin in the presence of glutathione, both strains were viable. There were 2.5 x 10⁸ (ml culture)^"1) colonies of Fragl (wild type) under these conditions (figure 4). However, the gshA mutant showed a far greater viability and there were 1.5 x 10⁹ (ml culture)^"1) colonies of MJF355 (gshA) (figure 4). The patterns of growth under these conditions are identical to those observed for AN2342 (wild type) and AN2343 (cydD). Both mutants display a far greater viability than their isogenic parents when challenged with benzylpenicillin in the presence of 2 mM glutathione. The morphology of MJF355 (gshA) after 16 h when grown under these conditions was comparable to when grown on LB alone (1 - 2 mm), whereas colonies of Fragl appeared as 'pinpricks'. EXAMPLE 14

Glutathione is transported by CydDC in an ATP-dependent manner

We have demonstrated that L-cysteine is a substrate of the CydDC transporter. Uptake into everted membrane vesicles of AN2342 (wild type) was shown to be ATP- dependent and protonophore insensitive. In addition, the ATP analogue sodium orthovanadate inhibited transport, as expected from an ATP-dependent transporter. The ability of GSH to rescue phenotypes associated with periplasmic redox stress provides some evidence that it is also a substrate of CydDC. To directly test this, uptake of [³⁵S]glutathione into everted membrane vesicles of AN2342 (wild type) and AN2343 (cydD) was measured. The success of previous uptake studies using [³⁵S]cysteine, highlights the effective use ofthe membrane vesicle protocol employed by Ambudkar et al (32) to generate membrane vesicles with a predominantly everted sidedness, and therefore the same conditions were applied to generate everted membrane vesicles for use in this study. As in previous studies, to demonstrate that the everted vesicles support active transport, the ability of the vesicles to accumulate [^l Cjlactose in response to an energised membrane was measured. Everted vesicles prepared from cells grown in MOPS medium supplemented with glucose have a low rate of lactose transport. Therefore, everted vesicles were prepared from strain AN2342 grown in MOPS medium supplemented with 40 mM lactose as described in Materials and Methods. Significant accumulation of [¹⁴C]lactose occurred only if D-lactate was added as an energy source (data not shown). Typical rates of [¹⁴C]lactose transport were 0.2 nmol min^"1 (mg protein)^"1 (data not shown). The addition of the proton ionophore, CCCP, abolished transport, demonstrating that accumulation of [¹⁴C]lactose was dependent upon Δp (data not shown). When investigating GSH transport into everted membrane vesicles, the assay constituents were pre-equilibrated at 30 °C before transport was initiated by the addition of 10 mM ATP. No uptake of [³⁵S]GSH was observed in the 5 min prior to the addition of ATP in vesicles of AN2342 (Fig. 10). Upon addition of ATP, however, [³⁵S]GSH uptake was rapid with maximal uptake occurring over a 5 min period following ATP addition. The maximum rate of [³⁵S]GSH uptake observed under the conditions tested was 3.8 nmol min^"1 (mg protein)^"1. The uptake of [³⁵S]GSH reached a maximal level 5 min after the addition of ATP and apparent saturation was observed (Fig. 10). No [³⁵S]GSH uptake was observed in vesicles of AN2343 (cydD) before or after the addition of ATP. The CydDC transporter is an ABC-type transporter (9) and is expected to derive the energy for transport directly from ATP. Therefore, this was tested experimentally with transport inhibitors. The addition of CCCP (2 μM) had no discernible effect upon the uptake of [³⁵S]GSH by vesicles of AN2342 (orβ99) (Fig. 10), and the rate of [³⁵S]GSH uptake, was approximately 3.3 nmol (mg protein)^"1. There was no [³⁵S]GSH uptake into everted vesicles of AN2343 (cydD) in the presence of CCCP (Fig. 5B). Incubation of everted membrane vesicles of AN2342 (wild type) in the presence of sodium orthovanadate (an inhibitor of ABC-transport systems), abolished transport completely such that the rate was indistinguishable to that observed with everted vesicles of AN2343 (cydD) (Fig. 10). The combined data suggest that [³⁵S]GSH is a substrate of the CydDC transporter and accumulation into everted membrane vesicles is ATP-dependent, ionophore insensitive.

EXAMPLE 15

Oxidised glutathione is not a substrate of CydDC

The ability of GSH to correct phenotypes associated with periplasmic redox stress such as motility and resistance to benzylpenicillin, are more consistent with that of the antioxidant. For example, Dailey and Berg (62) demonstrated that motility defects in a dsbB mutant could be corrected by the exogenous addition of cystine. Therefore, it may be the oxidised form of glutathione that is transported by CydDC, and the [³⁵S]GSH shown to be taken up into everted membrane vesicles (Fig. 10) may in fact be GSSG that was formed as a result of GSH oxidation during the transport assay. Therefore transport assays to measure GSSG uptake by CydDC were performed. The [³⁵S]GSH obtained from Amersham Pharmacia is dissolved in an aqueous solution containing 10 mM DTT, which maintains the GSH in a reduced state. Therefore, to obtain GSSG from GSH, the DTT was first removed by solvent extraction with ethyl acetate as described. Upon removal of the DTT, the GSH was oxidised by exposing to air for 24 h. To determine if oxidation was complete, a DTNB assay was performed as described in Materials and Methods. DTNB undergoes oxidation in the presence of sulfhydryl groups and is therefore useful in monitoring the redox state of glutathione. A cold GSSG solution identical in concentration to that of the [³⁵S]GSH solution was used in the assay as a control to determine the extent of [³⁵S] glutathione oxidation. The transport assay conditions to determine [³⁵S]GSSG uptake into everted membrane vesicles were identical to those employed for [³⁵S]GSH. No uptake of GSSG into everted membrane vesicles of AN2342 was observed, even after the addition of 10 mM ATP (Fig. 11). Conversely, uptake of [³⁵S]GSH was rapid after the addition of 10 mM ATP (Fig. 11). Therefore, CydDC can support uptake of [³⁵S]GSH but not [³⁵S]GSSG.

EXAMPLE 1

A gshA mutant assembles cytochrome d and exogenous GSH cannot restore a functional bd oxidase in a cydD mutant

A clear phenotype of a cydD mutant is the inability to produce a functional cytochrome bd. We have demonstrated that GSH is a substrate of the CydDC transporter, and therefore it may be required in cytochrome bd assembly. To test this, cytochrome bd . assays were performed on strain MJF355 (gshA). It would be expected that this strain is deficient in cytochrome bd if indeed GSH is required for cytochrome d assembly. Strains Fragl (wild-type) and MJF355 (gshA) were grown aerobically and difference absorbance spectra (CO + reduced minus reduced) were recorded on whole cell samples. Spectra of the wild-type strain showed a band at 644 nm, corresponding to the carbonmonoxy form of cytochrome d. The levels of cytochrome bd were recorded at approximately 0.05 nmol mg protein^"1. Identical conditions were used to generate CO + reduced minus reduced spectra of the gshA. Identical spectral signals to those obtained for Fragl were recorded, indicating the presence of cytochrome d. In addition, the amount of cytochrome d was identical to that produced by Fragl (0.05 nmol mg protein^{" l}). Thus, there is no difference in cytochrome d assembly between Fragl (wild type) an MJF355 (gsA-4).

To investigate the role of GSH in cytochrome d assembly further, it was determined if the addition of exogenous GSH could restore a functional bd oxidase to a cydD mutant. AN2342 (wild type) and AN2343 (cydD) were grown up aerobically at 37 °C. To a growing culture of AN2343 (cydD), one of the following GSH concentrations was added: 0.1, 0.25, 0.5, 1, and, 2 mM. The GSH solutions ψere made up fresh prior addition to the culture to ensure oxidation was minimised. The use of DTT to maintain reduced glutathione was avoided as it has been previously reported that AN2343 is sensitive to this reducing agent. The cultures were allowed to grow until stationary phase before being harvested. The OD₆oo of AN2343 containing GSH above 0.1 mM, grew very poorly and^' at 2 mM GSH, growth was inhibited to such a degree that 2.5 1 of

^■"i culture were required to gain enough material to perform the cytochrome d assays. The (CO + reduced) minus reduced difference spectra were carried out as described in Materials and Methods. The spectra recorded for AN2342 (wild type) revealed a band at 644 nm, corresponding to the carbon onoxy form of cytochrome d, as described before. The conditions under which the cells were grown ensured the production of cytochrome d. Spectra of AN2343 (cydD) grown without the addition of GSH produced no band at 644 nm, indicative that cytochrome d is absent. In addition, the spectra of AN2343 to which GSH had been added were identical to this. Therefore no cytochrome d was observed in any ofthe samples to which GSH had been added.

REFERENCES

1. Ingledew, W. J. and Poole, R. K. (1984) Microbiol. Rev. 48, 222-271

2. Gennis, R. B. and Stewart, V. (1996) in Escherichia coli and Salmonella

typhimurium: Cellular and Molecular Biology, 2nd edition (Niedhardt, F. C, Curtis, R., Ingraham, J. R., Lin, E. C. C, Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M. and Umbarger, H. E., ed.) pp. 217-261, American Society for Microbiology, Washington, DC.

3. Jϋnemann, S. (1997) Biochim. Biophys. Ada 1321, 107-127

4. Poole, R. K. and Cook, G. M. (2000) Adv. Microbial Physiol. 43, 165-224

5. Way, S. S., Sallustio, S., Magliozzo, R. S. and Goldberg, M. B. (1999) J. Bacteriol.

181, 1229-1237

6. Endley, S., McMurray, D. and Ficht, T. A. (2001) J. Bacteriol. 183, 2454-2462

7. Georgiou, C. D., Fang, H. and Gennis, R. B. (1987) J. Bacteriol. 169, 2107-2112

8. Poole, R. K., Williams, H. D., Downie, J. A. and Gibson, F. (1989) J. Gen.

Microbiol 135, 1865-1874

9. Poole, R. K., Hatch, L., Cleeter, M. W. J., Gibson, F., Cox, G. B. and Wu, G. (1993)

Mol. Microbiol 10, 421-430

10. Poole, R. K., Gibson, F. and Wu, G. (1994) FEMSLett. Ill, 217-224

11. Saurin, W., Hofhung, M. and Dassa, E. (1999) J. Mol. Evol 48, 22-41

12. Goldman, B. S., Gabbert, K. K. and Kranz, R. G. (1996) J. Bacteriol. 178, 6338- 6347

13. Delaney, J. M., Ang, D. and Georgopoulos, C. (1992) J. Bacteriol: 174, 1240-1247

14. Siegele, D. A., I lay, K. R. C. and Imlay, J. A. (1996) J. Bacteriol. 178, 6091-

6096

15. Cook, G. M. and Poole, RK (2000) Microbiology 146, 527-536

16. Silhavy, T. J., Berman, M. L. and Enquist, L. W. (1984) Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 17. Dassler, T., Maier, T., Winterhalter, C. and Bock, A. (2000) Mol. Microbiol. 36,

1101-1112

18. Miller, J. H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY

19. Stewart, V. and Parales, J. (1988) J. Bacteriol. 170, 1589-1597

20. Willis, R. C, Morris, R. G., Cirakoglu, C, Schellenberg, G. D., Gerber, N. H. & Furlong, C. E. (1974) Arch. Biochem. Biophys. 161, 64-75

21. Brickman, E. & Beckwith, J. (1975) J. Mol. Biol. 96, 307-316

22. Michaelis, S., Inouye, H., Oliver, D. & Beckwith, J. (1983) J. Bacteriol. 154, 366-

374

23. Qi, S.-Y., Moir, A. J. G. & O'Connor, C. D. (1996) J. Bacteriol. 178, 12032-12038

24. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448

25. Gaitonde, M. K. (1967) Biochem. J. 104, 627-633

26. Kalnenieks, U., Galinina, N., Bringer-Meyer, S. and Poole, R. K. (1998) FEMS

Microbiol. Lett. 168, 91-97

27. Kita, K., Konishi, K. and Anraku, Y. (1984) J. Biol Chem. 259, 3368-3374

28. Bamford, V. A., Angove, H. C, Seward, H. E., Thomson, A. J., Cole, J. A., Butt, J.

N., Hemmings, A. M. and Richardson, D. J. (2002) Biochemistry 41, 2921-2931

29. Markwell, M. A., Haas, S. M., Bieber, L. L. and Tolbert, N. E. (1978) Anal.

Biochem. 87, 206-210

30. Butler, J., Spielberg, S. P. and Schulman, J. D. (1976) Anal Biochem. 75, 674-675

31. Anderson, M. E. (1985) Meth. Enzymol 113, 548-555

32. Ambudkar, S.V, Zlotnick, G.W. and Rosen, B.P. (1984) J. Biol. Chem. 259, 6142- 6145 33. Davidson, A. L. (2002) J. Bacteriol 184, 1225-1233

34. Blattner, F. R., Plunkett, G., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B. and Shao, Y. (1997) Science 277, 1453-1462

35. Pugsley, A. P. (1993) Microbiol. Rev. 57, 50-108

36. Andrews, j. C.,'Blevins, T. C. and Short, S. A. (1986) J. Bacteriol. 165, 428-433

37. Bonthon, D. T. (1990) Gene 91, 101-105

38. Gooley, A. A. and Packer, N. H. (1997) Proteome Research: New Frontiers in Functional Genomics pp. 63-69, Springer, Berlin

39. Yi , H. H. and Villarejo, M. (1992) J. Bacteriol. 174, 3637-3644

40. Kustu, S. G., McFarland, N. C, Hiu, S. P., Esmon, B., and Ames, G. F. (1979) J.

Bacteriol 138, 218-234

41. Boos, W. and Lucht, J. M. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C, Curtiss, R., Ingraham, J. L., Lin, E. C. C,

Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M. and Umbarger, H. E.) Second Edition, pp. 1175-1209. American Society for

Microbiology Press, Washington, D.C.

42. Goldman, B. S., Gabbert K. K. and ranz, R. G. (1996) J. Bacteriol. 178, 6348-

6351

43. Siegele, D. A. and Kolter, R. (1993) Genes Dev. 7, 2629-2640

44. Blair, D. F. (1995) Ann. Rev. Microbiol. 49, 489-522

45. Harshey, R. M. (1994) Mol. Microbiol. 13, 389-394

46. Crooke, H. and Cole, J. (1995) Mol. Microbiol. 15, 1139-1150 47. Bardwell, J. C., McGovem, K. and Beckwith, J. (1991) Cell 67, 581-589

48. Bardwell, J. CA, Lee, J.-O., Jander, G., Martin, N, Belin, D. and Beckwith, J. (1993) Proc. Natl. Acad. Sci. USA 90, 1038-1042

49. Dartigalongue, C, Nikaido, H. and Raina, S. (2000) EMBO J. 19, 5980-5988

50. Dawson, R. M. C, Elliott, D. C, Elliott, W. H. and Jones, K. M. (1986) Data for Biochemical Research, Third Edition, Clarendon Press, Oxford

51. Thδny-Meyer, L. (1997) Microbiol. Mol. Biol. Rev. 61, 337-376

52. Krantz, R., Lill, R., Goldman, B., Bonnard, G. and Merchant, S. (1998) Mol.

Microbiol. 29, 383-396

53. Page, M. D., Sambongi, Y. and Ferguson, S. J. (1998) Trends Biochem. Sci. 23,

103-108 ^'

54. Stewart, E. J., Katzen, F. and Beckwith, J. (1999) EMBO J. 18, 5963-5971

55. Cung, J., Chen, T. and Missiakas, D. (2000) Mol. Microbiol. 35, 1099-1109

56. Sambongi, Y. and Ferguson, S. J. (1994) FEBS Lett. 353, 235-238

57. Cook, G. M., Loder, C, Søballe, B., Stafford, G., Membrillo-Hernandez, J. and

Poole, R. K. (1998) Microbiology 144, 3297-3308.

58. Adler, L. W., Ichikawa, T., Hasan, S. M., Tsuchiya, T. and Rosen, B. P. (1977) J.

Supramol. Struc. 7, 15-27

59. Rensing, C, Fan, B., Sha ma, R., Mitra, B. and Rosen, B. P. (2000) Proc. Natl.

Acad. Sci. USA 97, 652-656

60. Nichόlls, D. G. and Ferguson, S. J. (2002) Bioenergetics 3, Academic Press, London

61. Cook, G. M., Cruz-Ramos, H., Moir, A. J. G. and Poole, R. K. (2002) Archiv. Microbiol, in the press

62. Dailey, F. E. and Berg, H. C. (1993) Proc. Natl. Acad. Sci. USA 90, 1043-1047.

Claims

1. A bacterial cell wherein said cell is genetically modified which modification is the transformation of said cell with a nucleic acid molecule wherein said nucleic acid molecule encodes a polypeptide with the specific enzyme activity associated with the CydDC transporter and further wherein said enzyme activity is overexpressed when compared to a non-transformed reference bacterial cell of the same species.

2. A cell according to Claim 1 wherein said nucleic acid molecule is selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented by Figure 13a andl3b; ii) . a nucleic acid molecule which hybridises to the nucleic acid molecule in (i) and which has the enzyme activity associated with the CydDC transporter; iii) a nucleic acid molecule consisting of a nucleic acid sequence which is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii) above.

3. A cell according to Claim 2 wherein said nucleic acid molecule hybridises under stringent hybridisation conditions to the sequence presented in Figure 13a and 13b.

4. A cell according to Claim 2 or 3 wherein said nucleic acid molecule consists ofthe nucleic acid sequence presented in Figure 13a and 13b.

5. A cell according to any of Claims 1-4 wherein said cell over-expresses said enzyme activity by at least two-fold when compared to a non-transformed reference cell ofthe same species.

6. A cell according to any of Claims 1-4 wherein said enzyme activity is over expressed at least 3-fold; 4-fold; 5-fold; 6-fold; 7-fold; 8-fold; 9-fold; or at least 10- fold.

7. A cell according to any of Claims 1-4 wherein said enzyme activity is over expressed at least 20-fold; 30-fold; 40-fold; or at least 50-fold.

8. A cell according to any of Claims 1-4 wherein said enzyme activity is over expressed by at least 100-fold.

9. A cell according to any of Claims 1-8 wherein said over-expression of enzyme activity is the provided by a variant gene which is modified by addition, deletion or substitution of at least one amino acid residue, and which has the activity of the CydDC transporter wherein said activity is enhanced when compared to an unmodified reference gene as represented by Figure 14a and 14b.

10 A cell according to any of Claims 1-9 wherein said bacterial cell is a Gram negative bacterial cell.

11 A cell according to Claim 9 wherein said cell is Escherichia coli.

12. A cell according to any of Claims 1-9 wherein said cell is a Gram positive bacterial cell.

13. A cell according to Claim 12 wherein said cell is ofthe genus Bacillus spp.

14. A cell according to Claim 13 wherein said cell is selected from the group consisting of: B. subtilis; B. licheniformis; or B. amyloliquefaciens.

15. A cell culture vessel comprising a cell according to any of Claims 1-14 and medium sufficient to support the growth of said cell.

16. A cell according to Claim 15 wherein said vessel is a fermentor.

17. A method for the manufacture a molecule comprising the steps: i) providing a vessel according to Claim 15 or 16; ii) providing cell culture conditions which facilitate the growth of a cell culture contained in said vessel; and optionally iii) isolating said molecule from said cell or said cells surrounding growth medium.

18. A method according to Claim 17 wherein said molecule is a polypeptide.

19. A method according to Claim 18 wherein said polypeptide contains a sulphur- containing amino acid.

20. A method according to Claim 17 wherein said molecule is a peptide.

21. A method according to Claim 20 wherein said peptide, is glutathione, or a structural variant of glutathione.

22. A method according to Claim 17 wherein said molecule is an amino acid.

23. A method according to Claim 22 wherein said amino acid is a sulphur containing amino acid.

24. A method according to Claim 23 wherein said amino acid is cysteine or cystine.

25. A method according to any of Claims 17-24 wherein said cell is a Gram positive bacterial cell.

26. A method according to any of Claims 17-24 wherein said cell is a Gram negative bacterial cell.