A Bacillus subtilis bglA gene encoding phospho-β-glucosidase is inducible and closely linked to a NADH dehydrogenase-encoding gene

Gene, 140 (1994) 85590 8 1994 Elsevier Science B.V. All rights reserved.

GENE

85

0378-l 119/94/$07.00

07725

A Bacillus subtilis bgZA gene encoding phospho+glucosidase is inducible and closely linked to a NADH dehydrogenase-encoding gene (Cloning;

sequencing;

gene mapping;

Northern

blotting;

mutations;

complementation

Jianke Zhang and Arthur Aronson Department

qf Biological Sciences, Purdue University, West Lafayette, IN 47907, USA

Received by J.A. Hoch: 7 June 1993; Revised/Accepted:

14 October/l4

October

1993; Received at publishers:

25 November

1993

SUMMARY

A 2.7-kb Hind111 fragment from Bacillus subtilis contains an open reading frame (ORF) encoding a protein with homology to an Escherichia coli phospho+-glucosidase B (PBG B). The B. subtilis gene was induced by aromatic P-glucosides, as judged by Northern hybridization and could complement an E. coli bglB mutant. Immediately downstream from this B. subtilis bglA gene, there was a partial ORF on the opposite strand which encoded a polypeptide with extensive homology to NADH dehydrogenase from an alkalophilic Bacillus. These genes were mapped to 340” between hut and gnt on the B. subtilis chromosome. Disruption of these genes by insertion of a neomycin-resistanceencoding gene (neo) did not result in any phenotypic changes comparable to those found in E. coli mutants.

INTRODUCTION

The Escherichia coli bglB gene encodes a PBG B which hydrolyzes phosphorylated P-glucosides such as salicin and arbutin (Schaefler and Malamy, 1969; Schnetz et al., 1987). The bgl operon in E. coli is comprised of three genes, bglG, bglF and bglB (Schnetz et al., 1987). The bglG gene encodes an RNA-binding protein that prevents termination of transcription in the presence of P-glucosides. The product of the bglF gene is a phosphoenolpyruvate-dependent enzyme I&, which simultaneously transports and phosphorylates arbutin and

Correspondence

to: Dr. Jianke

Zhang,

Department

of Biochemistry,

Purdue University, West Lafayette, IN 47907, USA. Tel. (l-317) 494-7966; Fax ( l-3 17) 494-7897; e-mail: [email protected] Abbreviations: aa, amino acid(s); B., Bacillus; bg/A, B. subtilis gene encoding PBG; bg/B, Escherichia coli gene encoding PBG B; bp, base pair(s); CCC, Genetics Computer Group (Madison, WI, USA); kb, kilobase or 1000 bp; NADH, reduced form of nicotinamide-adenine dinucleotide; ndh, NADH dehydrogenase (Ndh)-encoding gene; neo, neomycin-resistance-encoding gene; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PBG, phospho-Bglucosidase; tsp,transcription start point(s); wt, wild type; YAC, yeast artificial chromosomes. SSDI 0378-l

119(93)E0721-0

salicin (Houman et al., 1990). The E. coli bgl operon is cryptic in the wild-type (wt) strain but can be activated by insertion of either IS1 or IS5 into a region 78 to 125-bp upstream from the transcription start point (tsp) or by base substitutions in the CAP-CAMP binding site (Reynolds et al., 1981; 1986). There are two other cryptic operons in E. coli, ccl (Parker and Hall, 1990) and USC (Hall and Xu, 1992) which are also involved in the utilization of salicin, arbutin and cellobiose. Fortuitously, a B. subtilis gene with considerable sequence homology to the E. coli bglB gene was cloned. In addition, the C terminus of a closely linked ORF was found on the opposite strand which encodes a protein with considerable homology to an alkalophilic Bacillus NADH dehydrogenase. The relation of the bgl-like gene to the E. coli bglB was studied by examining requirements for gene expression, rescue of an E. coli mutant and the phenotypic effects of a null mutation. EXPERIMENTAL

AND DISCUSSION

(a) Gene cloning A 17-nt degenerate oligo probe designed on the basis of the aa sequence of a dodecapeptide isolated from the

86 spore coat insoluble

fraction

of B. subtilis JH642 (Zhang

et al., 1993) hybridized to a 2.7-kb Hind111 fragment a digest of B. subtilis JH642 DNA under conditions ilar to those

described

previously

(Zhang

Another

from sim-

et al., 1993).

This 2.7-kb fragment was cloned into M13mp19 in both orientations and overlapping deletions were generated by exonuclease Madison,

III WI,

with USA).

the

Erase-a-Base

These

were

GenBank

of 479 established

aa.

A search

that the deduced

ORF

was found

immediately

from an alkalophile, 1991). The alignment showed

85.5% identity

Bacillus sp. strain YN-1 (Xu et al., of 372 aa from the partial sequence with no gaps (Fig. 2B).

sequenced

with

a

for

homology

in

aa sequence

was

(b) Northern hybridization B. subtilis JH642 cells were grown in minimal (Spizizen

et al., 1958) with either glucose, arbutin,

medium or sali-

tin as the sole carbon source (Schnetz et al., 1987). Total RNA was extracted as described by Wu et al. (1989) and

very similar to that of the bglB gene of E. coli (Schnetz et al., 1987) with 49% identity and more than 80% sim-

DNA was removed by treatment DNase I (Boehringer-Mannheim,

ilarity

at 37°C for 1 h. RNA (30 ug) was fractionated

(Fig. 2A).

down-

kit (Promega,

Sequenase Kit (US Biochemical, Cleveland, OH, USA; Fig. 1). There is one ORF (nt 76 to 1515) encoding a polypeptide

partial

stream on the opposite strand. The deduced aa sequence is very homologous to that of a NADH dehydrogenase

with ribonuclease-free Mannheim, Germany) in a 2.2

Fig. 1. Nucleotide sequence of a 2749-bp fragment containing the bgl.4 gene and the C terminus of an ndh gene from 8. subtilis JH642 and the deduced aa sequences. Numbering of the partial aa sequence deduced from ndh is arbitrary and the first aa (alanine) could correspond to about aa 130 to 140 in the complete sequence assuming that it is similar in size to the NADH dehydrogenase from an alkalophilic Bacillus (see Fig. 2B). The directions of transcription are indicated with vertical arrows on the left. The presumptive ribosome-binding site for the b&l gene is doubly overlined. Inverted broken arrows identify two potential transcription termination signals. Two EcoRI sites used in the construction of a null mutant (see section d) are also marked. Asterisk (*), stop codon. The GenBank accession No. is Ll9710.

87 This

probe

weakly

hybridized

strongly

to a 1.5-kb mRNA

to a 2-kb

from cells grown

mRNA

and

on arbutin

or salicin (Fig. 3, lanes 2 and 3), but barely to a 2-kb mRNA from cells grown on glucose (Fig. 3, lane 1). It appears that this gene is inducible by specific substrates. There is a Rho-independent-like terminator immediately downstream from the bglA coding region (Fig. l), so the 1.5-kb mRNA immediately

could be produced upstream

from a weak promoter

from the bglA coding

region. The

more prevalent 2-kb mRNA could be transcribed from a stronger promoter about 500-bp upstream from the bglA start

codon,

so there

may be a second

gene upstream

from bglA. (c) Genetic complementation In order to confirm that this bglA gene encodes a PBG, a complementation test was carried out with an E. coli bglB mutant strain, NS376 (bglR bglB::TnS). The 2.7-kb Hind111 fragment was cloned into pUC19 to produce plasmid pJZO(+) such that the lac promoter was utilized to express the B. subtilis bglA gene. This fragment was also cloned in the opposite orientation in plasmid pJZO(-) as a control. The plasmid constructs were transformed into E. coli NS376 by selecting for ampicillin resis-

12

Fig. 2. Amino-acid sequence alignments between PBG of B. subtilis (Bs-PBG) and PBG B of E. di (EC-PBG) (A), as well as between the C terminus of the NADH dehydrogenase (Ba-Ndh) from an alkalophile, Bacillus sp. strain YN-1 and that (Bs-Ndh) from B. subtilis JH642 (B). Numbering of the Ba-Ndh is from Xu et al. (1991). The alignment was generated with the FASTA program in the GCG software package. The relationships between aa pairs: (I). identical; (:), highly conserved (Devereux et al., 1984). Dashes indicate gaps introduced for optimal alignment. Asterisk (*) marks the stop codon.

M formaldehyde-l% agarose gel (Miller, 1987) and blotted onto 0.45-pm BA-S 85 reinforced nitrocellulose (Schleicher&Schuell, Keene, NH, USA) using a VacuGene vaccum blotter system (Pharmacia, Piscataway, NJ, USA). A 0.65-kb HindIII-EcoRI fragment from the 5’ end of the 2.7-kb fragment (Fig. 1) was labeled with [a-32P]dCTP with a Multiprime DNA labeling kit (Boehringer-Mannheim) and used as a probe in Northern hybridization (Mahmoudi and Lin, 1989).

3

4

2kb-

1.5kb-

-1.15kb .@

-0.65kb

Fig. 3. Northern hybridization of B. subrilis RNA to a bglA gene probe. RNA (30 pg of each) was fractionated in a 2.2 M formaldehyde-l % agarose gel. transferred to nitrocellulose, and hybridized to a 0.65-kb HindHI-EcoRI fragment from the N terminus of the bglA gene (see section b). Total RNA was prepared from B. subtilis JH642 cells grown in liquid minimal medium containing 0.5% glucose (lane I). arbutin (lane 2), or salicin (lane 3), or from strain 5230 [ a(bgl.4~ndh)::neo] grown on salicin (lane 4). RNA size standards (0.24 to 9.49 kb) from BRL (Gaithersburg, MD, USA) were used.

88

tance

(50 yg/ml).

MacConkey

Transformants

agar containing

1987). Cells containing

plasmid

nies, indicating

that there

those containing

plasmid

dition,

pJZO(-)

was salicin

pJZO(-)

of its deduced and its ability

product

plasmid

pJZO(+) agar

but

white. In adbut not

medium

with

to the E. coii PBG

by arbutin

B (Fig. 2A)

an E. co& bglB mutant, and salicin

subtilis gene is likely to encode

as

(Fig. 3), this B.

location

of the bglA gene was deter-

mined by hybridization of a radiolabeled 1374-bp EcoRI fragment (nt 648 to 2022; Fig. 1) to an ordered collection of B. subtilis

DNA

segments

cloned

in yeast

artificial

chromosomes (YAC) covering more than 98% of the genome (Azevedo et al., 1993). A nylon filter onto which of these YAC clones

hybridized

to the probe

had been

as described

dot-blotted

was

~Mahmoudi

and

Lin, 1989). This 1374-bp probe hybridized strongly with YAC clone, 11-237, which contained a 180-kb segment of the B. subtilis chromosome including the gnt, glv-A, &a and purA markers. There was also weaker hybridiza-

a PBG and is, therefore,

bglA.

designated

(e) Gene mapping The chromosomal

DNAs

source. Based on the similarity

to complement

well as induction

onto et al.,

fermentation

remained

grew on minimal

salicin as the sole carbon

streaked

pJZO(+) formed red colo-

NS376 cells containing

plasmid

were

0.5% salicin (Schnetz

tion with an overlapping clone, 1332, which contains a 190-kb fragment of the B. subtilis chromosome including the hut marker

(d) Mutant construction and phenotypic effects A bglA null mutant was constructed by replacing segment of the coding region with a neomycin-resistanceencoding

gene (neo). Plasmid

pBR322 was digested

a

with

Hind111 + EcoRI and mixed with the 2.7-kb Wind111 fragment

containing

the bglA gene. Blunt

ends were gener-

ated by Sl nuclease treatment and the fragments were ligated with T4 ligase. The original EcoRI site of pBR322 was destroyed in the resulting plasmid, pJZ1. This plasmid was digested with EcoRI to remove a 1.37-kb fragment from the 2.7-kb insert (two sites at nt 648 and 2022, Fig. 1) and then ligated

with a 1.3-kb EcoRI

from plasmid

which contains

sette (Itaya 288 codons gene. Plasmid

pBEST502

fragment

a neo gene cas-

et al., 1989). In the resulting pJZ2 plasmid, had been deleted from the 3’ end of the bglA pJZ2 was linearized

with SphI, transformed

into B. subtilis JH642 strain (Anagnostopoulos and Spizizen, 1961), and a transformant, 5230, selected on L-agar plus neomycin (5 ng/ml). EcoRI-digested chromosomal

DNA from this strain

and the wt JH642 strain

were fractionated in a 0.8% agarose gel and blotted to nitrocellulose. The radiolabeled 1.37-kb EcoRI fragment (nt 648 to 2022, Fig. 1) hybridized

to a 1.37-kb band from

DNA of the wt strain, but not to DNA from strain 5230 ~unpublished results). This result confirmed that a double crossover had occurred and that the 1.3-kb neo gene cassette had replaced the 1.37-kb mutant strain 5230. In addition,

EcoRI fragment in the Northern hybridization

revealed that truncated bglA mRNAs ( 1.15 and 0.65 kb) were produced by the mutant (Fig. 3, lane 4). This mutant strain still grew on a minimal medium with 0.5% arbutin or salicin (Schaefler and Malamy, 1969; Schnetz et al., 1987). There appears to be only one copy of the bglA gene in B. subtilis JH642, as judged by Southern hybridization (unpublished results) so there must be different ~-glucosidases in B. subt~lis which utilize these aromatic @glucosides.

(Azevedo

et al., 1993). It is unlikely

that

there are different genes on these two YAC clones hybridizing to this probe. As mentioned above, this probe hybridized only to a single band from the wt or bgfA mutant chromosomal DNA digested with different restriction enzymes indicating that the bg/A gene is present in both YAC clones in the region of overlap. The bglA gene is located, therefore, approximately at 340” between hut (335”) and gnt (344’) on the chromosome (Piggot, 1989). Interestin~y, a gene designated bgl which encodes a l&glucanase was also mapped in this region close to hut (334”; Borriss et al., 1986). (f ) The ndh gene NADH dehydrogenase catalyzes the electron transfer from NADH to the respiratory chain. There are two distinct species of NADH dehydrogenase in the E. coli cytoplasmic membrane. NADH dh-I is a multi-subunit complex and reacts with NADH as well as deaminoNADH (reduced nicotinamide hypoxanthine dinucleotide). NADH dh-II consists of a single polypeptide and reacts exclusively with NADH (Mutsushita et al., 1987). The NADH dehydrogenase isolated from an alkalophile, Bacillus sp. strain YN-1, is an NADH dh-II type dehydrogenase loosely bound to the cytoplasmic membrane and it specifically oxidizes NADH but not NADPH (Xu et al., 1991). The dh gene of B. subtilis JH642 identified in this work probably encodes a NADH dh-II type dehydrogenase since the deduced aa sequence shares extensive homology with the NADH dh-II dehydrogenase from the alkalophilic species (Fig. 2B). A ndh mutant of E. coli grows poorly with either succinate or glucose as a sole carbon sources. The mutant is unable to grow with mannitol as a sole carbon source presumably because the conversion of mannitol l-phosphate to fructose 6-phosphate generates one molecule of NADH which is not reoxidized in the subsequent metabolism of fructose to lactate (Young and Wallace,

89 1976). The C terminus of the B. subtilis JH642 ndh gene (131 codons) was coincidentally deleted in strain 5230

Steinmetz

because of its closeness to the bglA gene, but this strain still grew on minimal medium with mannitol as the sole

and data base searches were done with the GCG software package in the Purdue AIDS Research Center which is

carbon

supported

source.

nicating

sized

for providing unpublished

by NIH

in

the

grant

Purdue

Macromolecular

(g) Other possible bgl-like gene(s) in B. subtifis

B. subtilis strains and for commuresults.

Protein

sequence

A127713. Oligos University

analyses

were synthe-

Laboratory

for

Structure.

The lack of phenotypic effects in strain 5230 containing mutations in both the bglB and ndh genes which were anticipated from the results with E. coli, indicates that in B. subtilis there are other genes which overlap the functions of these two genes. Recently, another E. coli bglB homologue

was isolated

cated between

from B. subtilis which was lo-

hut and gnt on the chromosome

and the

deduced aa sequence is similar to but clearly different from the PBG A identified in this work (D. LeCoq and M. Steinmetz, personal communication). We made a double mutant by phage PBSl-mediated transduction (Hoch, 1991) of the neo marker of 5230 into a strain which contains a silent mutation in this second bgl-like gene (provided by Dr. M. Steinmetz). This double mutant still grew on minimal medium with salicin as the sole carbon source indicating that there is likely to be at least one other gene encoding an enzyme with similar substrate specificity. It is interesting that these functionally related bgl genes are all clustered in the same region of the genome, perhaps due to gene duplication. (h) Conclusions (1) A 2.7-kb Hind111 fragment from B. subtilis contains a complete bgl.4 gene and on the other strand the C terminus of a gene which is 85% identical to a ndh gene from an alkalophilic Bacillus. These genes were mapped to 340” on the B. subtilis chromosome. (2) The sequence similarity of the B. subtilis bglA gene to the E. coli bglB gene and its ability to rescue an E. coli bglB strain indicate that this gene encodes a PBG. This gene is inducible in B. subtilis, in contrast to the cryptic bgl operon in E. coli. It is probably transcribed at least in part as a polycistronic mRNA so there may be other linked bgl genes as in E. coli. (3) There was no phenotypic effect resulting from a deletion of portions of the bglA and ndh genes, suggesting the existence of genes with overlapping functions.

ACKNOWLEDGEMENTS

J.Z. was supported by a Purdue Research Foundation Fellowship. We thank Dr. Andrew Wright (Tufts University) for providing us the E. coli bglB mutant strain NS376 and Dr. P. Serror for the nylon filter with the YAC clones. We also thank Drs. D. LeCoq and M.

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