Short sequence-paper

A cysteine desulphurase gene from the cellulolytic rumen anaerobeRuminococcus £avefaciens1

James Kirby 2;a, Frank Wright b, Harry J. Flint a;*a Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK

b BIOSS, Scottish Crops Research Institute, Invergowrie, Dundee, UK

Received 19 February 1998; revised 30 March 1998; accepted 2 April 1998

Abstract

A gene whose predicted product shows 40^50% sequence identity with the products of nifS genes from nitrogen-fixingbacteria was found downstream from a cellulase gene in a DNA fragment from the cellulolytic rumen anaerobe,Ruminococcus flavefaciens 17. The R. flavefaciens gene product released sulphur from L-cysteine when expressed inEscherichia coli, indicating that the R. flavefaciens NifS enzyme may play a role in sulphuration, perhaps, as in nitrogen-fixing bacteria, supplying sulphur to FeS proteins. Sequences hybridising with the R. flavefaciens 17 nifS-like gene were alsodetected in R. flavefaciens 007 and in R. albus SY3. ß 1998 Elsevier Science B.V. All rights reserved.

Keywords: Cysteine desulfurase; nifS ; Rumen; Cellulolysis ; (Ruminococcus £avefaciens)

The strictly anaerobic bacterium Ruminococcus £a-vefaciens plays an important role in the degradationof cellulose and other plant polysaccharides in therumen [1,2]. It produces multiple cellulases and xyla-nases which are thought to form a multi-enzymecomplex that may be similar to the cellulosomefound in Clostridium thermocellum [3^5]. The speciesowes its name to the production, during growth oncrystalline cellulose, of a yellow substance [6] thatmay play a role in adsorption to crystalline cellulose[7]. We report here the isolation of a cysteine desul-phurase gene from a region of genomic DNA thatlies downstream from a cellulase gene in R. £avefa-

ciens 17. This gene is related to nifS genes which were¢rst isolated from nitrogen-¢xing bacteria [8,9] butare now thought to play important roles also innon-nitrogen-¢xing bacteria [10,11]. This is the ¢rstisolation of such a gene from an obligately anaerobicbacterium.

A 17 kb fragment of R. £avefaciens 17 DNA waspreviously isolated in the bacteriophage VEMBL3clone CMC4 by its activity against carboxymethylcellulose [12]. Partial sequencing of the phage clonehas since shown that it contains the endA gene,which is now known to encode a multidomain cellu-lase of 759 amino acids that carries a sequence re-sembling dockerins [3] found in cellulosome-associ-ated polypeptides from cellulolytic Clostridium sp.[13]. Plasmid subclones of CMC4 were obtained inpUC18 in order to study regions that £ank the endAgene, and sequences were obtained using an ABI 373automated sequencer following synthesis of appro-

0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 0 6 2 - 4

* Corresponding author. Fax: +44 (1224) 716687;E-mail : h.£int@rri.sari.ac.uk

1 Sequence accession No. AJ003152.2 Present address: Genetics Department, Trinity College,

Dublin, Ireland.

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priate primers. One of these subclones, HS17, whichcarries a 1.7 kb insert and maps approx. 1.5 kbdownstream from the endA gene, was found to con-tain an open reading frame (ORF) on the samestrand as endA, encoding a nifS-type protein. The

R. £avefaciens 17 nifS gene consists of a 1188 bpORF encoding a polypeptide of 396 amino acid res-idues of predicted molecular mass 43.2 kDa. Theputative translational start codon is TTG, which isfound not infrequently in place of the more usual

Fig. 1. Multiple amino acid sequence alignment (Clustal V) of NifS-like proteins from Ruminococcus £avefaciens (RUMFL), Anabaenasp. (ANASP), Azotobacter vinelandii (AZOVI), Bacillus subtilis (BACSU), Rhodobacter sphaeroides (RHOSH) and Saccharomyces cere-visiae (YEAST). Accession numbers are given in the legend to Fig. 2. The ¢rst 94 residues of the yeast Nfsl product were not includedin the alignment. Stars indicate conserved residues and dots conservative substitutions.

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ATG codon at the start of genes in low G+C contentGram-positive bacteria [14^16]. The proposed ribo-some-binding site, AGGAGG, precedes the transla-tional start by 8 bp. Codon usage for the nifS gene issimilar to that for other genes from R. £avefaciens 17[3]. A second partial open reading frame of 39 resi-dues terminates at the putative nifS ribosome-bind-ing site and is predicted to encode a product thatshows sequence similarity with the C terminus ofRNAase H from bacteria and higher organisms.RNAase H speci¢cally degrades the RNA compo-nent in RNA-DNA hybrid molecules [17].

The predicted R. £avefaciens NifS product showedsigni¢cant sequence similarity to ten NifS-type pro-teins in database searches and a multiple alignmentwith ¢ve of these sequences is shown in Fig. 1. Con-served residues corresponding to the proposed cata-lytic cysteine, Cys325, and pyridoxal phosphate-bind-

ing lysine, Lys202, reported in Azotobacter vinelandii[18] are present in R. £avefaciens. A phylogenetic treeof published NifS-type proteins is shown in Fig. 2,based on an approximate maximum likelihood anal-ysis. Similar results were obtained for the neighbourjoining method (not shown). Somewhat surprisinglythe yeast NifS sequence falls into a signi¢cant clustertogether with the R. £avefaciens protein and the NifSprotein from another Gram-positive bacterium, Ba-cillus subtilis. Possible explanations are that the yeastnifS gene is of endosymbiotic origin, or was acquiredthrough horizontal gene transfer.

In order to test NifS function, the 1.7 kb fragmentcarrying the nifS gene was excised by cleavage withSalI and HindIII and reinserted into pUC18 in thecorrect orientation for expression from the lacZ pro-moter. This construct, NS1, was transformed intoEscherichia coli HB101 containing the plasmidpNM52 which harbours a lacIq repressor [19], ensur-ing that expression of nifS was tightly regulated bythe addition of isopropyl L-D-thiogalactopyranoside(IPTG). A 42 kDa protein corresponding to the pre-dicted size of the nifS gene product was detected bySDS-PAGE in NS1 cells grown in the presence of1 mM IPTG (Fig. 3). Sonicated extracts of NS1 cat-alysed the production of sulphide from cysteine in thepresence of 1 mM dithiothreitol, as described for

Fig. 2. Phylogenetic relationships of NifS-like proteins. The treeshown is an unrooted quartet puzzling tree (performed usingthe Puzzle programme within the PHYLIP package). Figuresrepresent the % support for the branching indicated (based on1000 steps). Distances were derived by the approximate maxi-mum likelihood method. The scale bar indicates amino acidsubstitutions per position. RHOSH, Rh. sphaeroides (accessionNo. Q01179); RHOCA, Rhodobacter capsulatus (Q07177);AZOVI, A. vinelandii (P05341); AZOCH, Azotobacter chroococ-cum (P23120); RUMFL, R. £avefaciens (AJ003152); YEAST,S. cerevisiae (P25374); KLEPN, Klebsiella pneumoniae(P05344); ANASP, Anabaena sp. (P12623). The sequence ofLactobacillus delbrueckii NifS (P31672) was not included in thisanalysis because it is shorter than the other gene productsshown.

Fig. 3. SDS-PAGE demonstrating production of a 42 kDa pu-tative NifS protein in NS1. Lane 1 contains sonicated controlcells harbouring pUC18 and pNM52; lane 2 contains sonicatedcells harbouring NS1 and pNM52; M denotes molecular weightmarkers. Both NS1 and control cultures were induced with1 mM IPTG at inoculation.

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NifS from A. vinelandii [20] (Table 1). Activity wasfound to be maximal in NS1 at 37³C and pH 8.3^8.6(results not shown). Cysteine desulphurase activitiesin IPTG-induced NS1 cultures were around 150 timeshigher than in uninduced NS1 cultures and controlcultures, con¢rming that the activity arose from theR. £avefaciens nifS gene (Table 1). A yellow colourwas observed in cell pellets from E. coli cultures ex-pressing the R. £avefaciens nifS gene, a phenomenonalso observed when the A. vinelandii nifS gene wasexpressed in E. coli [20]. A fragment of the R. £ave-faciens 17 nifS gene was used to probe chromosomalDNA from rumen isolates of R. £avefaciens and R.albus. The R. £avefaciens nifS gene was found to hy-bridise at 65³C to R. £avefaciens strains 17 and 007,and R. albus strain SY3, suggesting the presence of anifS-like gene in all three strains (Fig. 4).

In A. vinelandii a single nifS gene was found to berequired for activity of three di¡erent nitrogenasesystems encoded by nif, vnf and anf genes, respec-tively [21,22]. The A. vinelandii NifS enzyme wasshown to be a pyridoxal phosphate-containing ho-modimer that catalyses the formation of L-alanineand elemental sulphur (So) from L-cysteine [20]. ANifS reactive cysteine residue, Cys325, was found toform a cysteinyl persulphide as an intermediate in thereaction which is the proposed sulphur donor in Fe-Score formation [18]. Subsequently, NifS was shownto catalyse activation of an apo form of the Fe pro-tein, supporting the proposal that NifS provides theinorganic sulphide necessary for in vivo formation ofthe nitrogenase metalloclusters [23]. More recently,genes homologous to nifS have been discovered inseveral non-nitrogen-¢xing bacteria, suggesting thatnifS may play a general role in iron-sulphur clusterassembly. A nifS homologue from B. subtilis is sus-pected to play a role in NAD biosynthesis, as muta-tions in the nifS gene resulted in a nicotinic acid-dependent phenotype [11]. A nifS-like gene in Sac-charomyces cerevisiae, named nfsl, was found to beone of only three essential genes on chromosome III.Nfsl was found to be essential for tRNA splicing, aNAD�-dependent process, but its precise functionhas not yet been ascertained [24]. NifS from A. vine-landii was shown to catalyse the in vitro assembly ofiron-sulphur clusters into E. coli SoxR protein, whichis essential for oxidative stress regulation [25]. Mean-while, a NifS homologue recently puri¢ed from E.coli contains pyridoxal phosphate as a cofactor andcatalyses the conversion of cysteine to alanine and So

(or H2S in the presence of DTT). The NifS homo-logue was shown to contribute to the formation of aFe4S4 cluster on the apoprotein of E. coli dihydroxy-acid dehydratase [10].

Both in nitrogen-¢xing bacteria and in B. subtilisthe nifS gene is located near genes encoding an en-zyme system which requires NifS for activity. In thecase of R. £avefaciens the proximity of the nifS geneto the endA gene suggests a possible link with thedegradation of cellulose. In some anaerobic bacteria,including ruminococci, cellulolytic activities mayshow susceptibility to oxygen [26,27] and it is con-ceivable that some step involved with cellulose deg-radation or with the uptake of degradation productsof cellulose is dependent on iron-sulphur proteins. It

Table 1Release of sulphide from cysteine by extracts of E. coli HB101cells carrying the R. £avefaciens 17 nifS-like gene

Plasmids Inducer H2S production(nmol min31 (mg protein)31)

pNM52, pUC18 3 2.01 ( þ 0.12)pNM52, pUC18 +IPTGa 1.95 ( þ 0.06)pNM52, NS1 3 2.34 ( þ 0.10)pNM52, NS1 +IPTGa 351 ( þ 13)

The gene is present in plasmid NS1 (see text).aCultures designated `+IPTG' were supplemented with isopropylthiogalactoside to 1 mM at inoculation to induce expressionfrom the lacZ promotor. Figures in parentheses refer to stand-ard errors.

Fig. 4. Hybridisation of the R. £avefaciens 17 nifS gene toHindIII-cut chromosomal DNA from R. £avefaciens strains 17and 007, and R. albus strain SY3 (lanes 1^3, respectively).

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may be of signi¢cance that the cellulosome of C.thermocellum contains iron (6 moles per mole of cel-lulosome based on a cellulosome molecular mass of4 MDa), but the location and form of this element inthe complex is not yet known [28]. The nature androle of the yellow pigment which accumulates in R.£avefaciens 17 cultures grown on crystalline celluloseis unclear, but there is at present no evidence tosuggest a link with sulphur deposition. On the otherhand it appears equally possible that the linkage ofnifS to a cellulase is coincidental and the closer link-age of nifS with rnh suggests that NifS might benecessary for another function altogether. The cellu-lar function of RNAase H in R. £avefaciens is notestablished, but in E. coli it is suspected to play a rolein DNA replication [29]. RNAase H itself containsno conserved Cys-X-X-Cys amino acid motifs indi-cative of iron-sulphur proteins, but other DNA- andRNA-binding proteins, such as endonuclease III,SoxR, and iron regulatory element-binding proteinhave been found to contain FeS clusters [30]. It isconceivable that an RNA- or DNA-binding proteinencoded by a linked gene requires NifS for activity.

In conclusion, this work demonstrates the presenceof a nifS-like gene in the non-nitrogen-¢xing strictanaerobe R. £avefaciens 17. The activity of thecloned gene product in E. coli suggests that the func-tion of NifS in R. £avefaciens 17 could involve theproduction of sulphur for the formation of iron-sul-phur clusters but little is known about iron sulphurproteins in this group of bacteria and the hypothet-ical target protein has not been identi¢ed. Indeed arole in sulphuration of other, unknown, molecules isnot ruled out, particularly since this is the ¢rst reportof a cysteine desulphurase gene from a strict anae-robe. NifS has more than one role to play in somespecies and may also turn out to have multiple func-tions in R. £avefaciens.

This work was supported by the Scottish O¤ceAgriculture, Environment and Fisheries Department.Molecular biology computing made use of the Dares-bury Seqnet facility (Daresbury, UK).

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