Tài liệu Báo cáo khoa học: Identi®cation and properties of type I-signal peptidases of Bacillus

Identi®cation and properties of type I-signal peptidases

of Bacillus amyloliquefaciens

Hoang Ha Chu, Viet Hoang*, Peter Kreutzmann², Brigitte Hofemeister, Michael Melzer

and JuÈ rgen Hofemeister

Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

The use of Bacillus amyloliquefaciensfor enzyme production

and its exceptional high protein export capacity initiated this

study where the presence and function of multiple type I

signal peptidase isoforms was investigated. In addition to

type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A.,

Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. &

van Dijl, J.M. (1995) Mol. Microbiol. 17, 621±631] and

SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim.

Biophys. Acta 1269, 64±68] which were previously identi®ed,

here we present evidence for two other Sip-like genes in

B. amyloliquefaciens. Same map positions as well as

sequence motifs veri®ed that these genes encode homologues

ofBacillus subtilis SipV and SipW. SipU-encoding DNA was

not found in B. amyloliquefaciens. SipW-encoding DNA

was also found for other Bacillus strains representing dif￾ferent phylogenetic groups, but not for Bacillus stearother￾mophilus and Thermoactinomyces vulgaris. The absence of

these genes, however, could have been overlooked due to

sequence diversity. Sequence alignments of 23 known Sip￾like proteins from Bacillus origin indicated further branching

of the P-group signal peptidases into clusters represented by

B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5

proteins, respectively. Each B. amyloliquefaciens sip(ba)

gene was expressed in an Escherichia coli LepBts mutant and

tested for genetic complementation of the temperature sen￾sitive (TS) phenotype as well as pre-OmpA processing.

Although SipS(ba) as well as SipT(ba) eciently restored

processing of pre-OmpA in E. coli, only SipS(ba) supported

growth at TS conditions, indicating functional diversity.

Changed properties of the sip(ba) gene disruption mutants,

including cell autolysis, motility, sporulation, and nuclease

activities, seemed to correlate with speci®cities and/or

localization of B. amyloliquefaciens SipS, SipT and SipV

isoforms.

Keywords: Signal peptidase I; Bacillus amyloliquefaciens;

protein secretion; E. coli; genetic complementation.

The principles of protein transport through membranes are

basically similar in eukaryotic and prokaryotic organisms

[1], although destinations of proteins are numerous in

eukaryotic cells but only few in bacterial cells, such as the

cytoplasmic membrane, periplasm, outer membrane, cell

wall, spore compartment, or the extracellular environment

[2±4]. The majority of export proteins are transported via

the Sec pathway by recognition and site-speci®c processing

[3±5]. These export proteins carry a particular N-terminal

leader (signal) peptide, which bears distinct domains (N, H

and C), that are distinguished by charge and hydrophobicity

pro®le [3±5]. The N- and H-regions are thought to interact

with the translocase machinery and to mediate membrane

insertion, whereas the C-region allows sequence-speci®c

cleavage by SPases and removal of the signal peptide from

the precursor (export) protein [4,6±8]. Minor differences

between individual signal peptides, speci®c properties of the

export protein precursor [5,7,9], as well as the speci®city of

distinct SPases [3,10,11] affects the processing of individual

or groups of export proteins. The B. subtilis genome

sequencing project [12] has enabled computer analysis to

predict that  166 proteins of the total B. subtilis proteome

contain a N-terminal signal peptide, characteristic for Sec

export protein precursors [4]. Several eubacteria and

archaebacteria possess only one type I SPase functioning

in Sec export protein processing [13]. However, B. subtilis

contains ®ve chromosomally encoded type I SPases, named

SipS, SipT, SipU, SipV, and SipW, respectively [4,5,14,15].

Multiple type I SPases were also found in Archaeoglobus

fulgidus [16], Streptomyces lividans [17], Bradyrhizobium

japonicum [18,19] and Staphylococcus aureus [20]. The

presence of a unique type I SPase (LepB in E. coli) was

shown to be essential for cell viability [21,22]. In contrast,

B. subtilis has ®ve Sip homologues, of which SipS as well as

SipT isoforms were shown to be essential for cell viability,

and have overlapping processing functions. Double mutants

Correspondence to J. Hofemeister, Institute of Plant Genetics and

Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben, D-06466,

Germany. Fax/Tel.: + 49 394825 138/241,

E-mail: [email protected]

Abbreviations: Ap, ampicillin; c.f.u., colony forming units; Cm, chlo￾ramphenicol; CWBP, cell wall bound proteins; Em, erythromycin;

pre-OmpA, OmpA precursor protein; Sip, signal peptidase protein;

SPase I, signal peptidase I (leader peptidase I); TS, temperature

sensitivity; IPTG, isopropyl thio-b-D-galactoside.

De®nitions: SipS(ba), SipS(bj), SipT(ba), SipV(ba) and SipW(ba) are

the products of the sipS(ba), sipS(bj), sipT(ba), sipV(ba), and

sipW(ba) genes of Bacillus amyloliquefaciens (ba) or Bradyrhizobium

japonicum (bj), respectively.

*Present address: George Beadle Center for Genetics, School of Bio￾logical Sciences, University of Nebraska, Lincoln, USA.

 Present address: Lower Saxony Institute for Peptide Research, Han￾nover, Germany.

(Received 29 May 2001, revised 5 November 2001, accepted 13

November 2001)

Eur. J. Biochem. 269, 458±469 (2002) Ó FEBS 2002