247 veviews

Bacillussubtifisandits relatives: molecularbiologicaland industrialworkhorses Cohn R.liar-wood The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry,

genetics and physiology of Gram-positive bacteria, and

particularly of sporulation and associated metabolism. Extensive knowledge 4 the molecular biology of 5. subtilis has led to the recent development of this tactYum as a host for the industrial production of heterologous proteins. Although difficulties have been encountered, these are being systematically addressed and overcome. Bacillcrsstrbtilis strain Marburg is the type strain of the genus Buci&rs, a group of bacteria with a long history of exploitation by mankind. The genus is one of the most ubiquitous and diverse, with representatives being found in the soil and assaciated water sources such as rivers, coastal waters and estuaries’. The meabolic diversity of these organisms has led to members of this group being used for a wide range of industrial processes, including the production ofhydrolytic (and other) enzymes. antibiotics, fine biochemicals and insecticides. One of the carlicst reported uses of Brmcillrls was the fermentation of soybeans into Natto. a traditional Japanese food, by BUC&J slrbtilis var. ndtt~~. The current annual consumpciom of Natto (of -?OO x 10” kg), taken together with the low level of reported incidence ofpathogenicity amongst strains of B. sltbtilisand the widespread use oftheir products and those of their close relatives in the food, beverage and detergent3 industries, has resulted to the granting of GRAS (generally regarded as safe) status to B. sltbtilis by the US Food and Drug Administration (FDA). Bacteria of the genus BQ&S are aerobic, endosporeforming Gram-positive rods. The genus has more than 50 validly described species’, but is heterogeneous. Taxonomic” and 16s rP.NA catalogling studies’ indicate that the genus might be more appropriately divided into four or five smaller genera. The heterogcneicy of the genus, particularly with respect to %GC content which can vary from J3% to 66% (B. srrtrilir is about 43X), is an important consideration for inrra-generic cloning since control regions and codon usage may not be optimal for high-level gene expression and protein fidelity. Bncilltrs strains have evolved a series of responses to cope with the fluctuation in nutrient supply that is a

0 1992. Elsetier Science Pubkhers Ltd (UK)

common characteristic of their natural environment. These responses, which are obse,yed in the laboratory towards rhe end of the exponential growth phase and in the stationary phase, include the onset of motility and associated chemotaxis, the induction ofhydrolytic enzymes (e.g. proteases, rarbohydrases) and the dcvelopment of competence to take up exogenous DNA”. If these transitory responses fail in enabling the organism to acquire sufticient growth-sustaining nunienrs, these organisms resort to forming endospores”. Three of these events - the development of comperence, the production of hydrolytic enzymes and sporulation ha\e been driving forces in the development of the molecular biology of B. s&lis. As a result. atnong the bacteria, our understanding of the biochemistry, physiology and genetics of B. srrb~ilisis sccona only to that of EsclgericlIioculi. Genetics and molecular biology of B. subtilis BaciHlrssrrbtilis has been a paradigm 2y.vrhe genetics of Gram-positive bacteria for more than 30 years. it has a particularly well-developed n>iuraI ;ransiom+ ation system thacpraved invaluable for gencnc manipulation and analysis in the pre-recombinant DNA tcchnology era. B. trrbrilic was the first non-pathogenic: microorganism to be transformed-more than 30 years ago by John Spizizen and his colleagues’. Spizizen used strain 168, a tryptophan auxotroph, and consequently this strain has formed the basis of nearly all subsequent genetical studies. Experimentally at least, strain 168 ot. B. s&&s may be considered the equivalent of strain K-12 of E. cob and strain LT2 of Sahonelln lypkimlmtitrrn. Tri2rufrmati0n andjifte-structure map@ig Transformation ofcompetent cells by chromosomal DNA is a relatively simple and reproducible procedures. Competence (the physiological state in which

Transducing yhage and lung-range mapping

-

-

Chromosome

amy E

m_..,p1 amyE

cal

mcs oriEc

( E b-amyE

bla

Chromosome

Figure1 Recombination events associated witi: Bacillus integrational vectors. Such plasmid vectors are incapable of replicating.autonomously in strains of Bacillus, but usually have an E. coli origin of replication (oriCc1 to permit their propagation, For example, (aI DNA is cloned between ttis end fragments lamyEA and damyEJ of the uamylase gene CamyE). Also located within this region is an antibiotic resistance gene (e.g. chloramphenicol resistaoce, cat) capable of being expressed in 8. subtilis. Selection for chloramphenicol resistance seiects recombinants that have undergone a double crossover event between the ends of the vector amyE gene, and the corresponding regions of the chromosomally encoded amyE gene which, as a result, is inactivated. This can be confirmed by the recombinant’s inability to degrade starch. !b) Recombination occurs by a single crossover event between homologous DNA on the vector and the host chromosome, and recombinants selected with (in this case) chloramphenicol. The homologous DNA used for recombination can be either (1) sequences introduced into the vector during its construction, in which case the vector is directed to a specific location on the host chromosome, or (2) random pieces of host chromosomal DNA ac&ir?d during the cloning event.

cc!ls are able to take up cxogcnous naturally

in 8. s&tiliv

towards

DNA) develops

tflc end OF cxpoxntial

growth, but may 5~ induced csperimcntalIy by specific growth regimes. At saturating DNA conccntmtions, single cells lllily t&C Up more tkin one nialcculc of DNA, a process referred to as congcssinn. Congression is an cstrenrcly valuablr technique for strain construction, particularly for markcni that arc not capable ofbcing sclcctcd direct&+:). At high DNA concentrations (>I p,g m-l), between one and four pcrccnt ofrransformants of an casiiy selected I?vrkcr will usually contain the unlinked sccood marker.

.I.... ..--________._

TIBTECH JULY1992 (VOL, 101

As a mapping tool, transformation is limited to finestructure mapping. in the absence of a conjugationbased, high frequency recombination (HG) genetransfer system, the gcncralized transducing phage PBS1 has been used for long-range mapping studies. Unlike its E. m/i equivalent (phage Pl), PBS1 is able to transduce extremely large fragments of chromosomal DNA; up to eight percent of the genome (corresponding to about 300 kb). PBS1 transduction is just as convenient as HG strains for mapping new markers, particularly when used in conjunction with mapping kits; sets of strains with auxotrophic lesions or sil+:nt transposon insertions located throughout the circular linkage niapl”J I. The absence of gcnctic systems cquivalcnt to the F-prime plasmids and A specialized transducing phagcs of E. cob, initially made compleilzentatioii analysis of B. strbtilh &f?icult, and various systems were developed to generate partial diploids (mcrodiploids)“. More rcccntly, specialized transducing phages have been idcntiticd and devclopcd, in many cases incorporating additional propcrtics that aid their selection and functionalityi~~l’. Extensive use has also been made of integrarionn! vectors that permit ths (ri>iniroduction of genes of interest at specific locations in the chromosome (Fig, l)s:‘.‘-. Thrsc vectors are basically E. m/i plasmids carrying markers that can bc selected in B. sr&ilic. In some of these vectors. fragments of 5. wbtilk chromosomal DNA are included to direct the gcncs to a specific chromosome locus by ‘quasi’-reciprocal recombination (Fig. la); in other cases, homologous cloned gcncs provide a site for .Campbcll-type’ integration events (Fig. lb). Integration vectors have also been used to generate mutants and for studying gene expression.

The CUITC’III liakag: map of B. srrbti!is 168 (Ref. 16) contains about 700 gcnctic loci, as compared with 1400 loci for I+?.n>/i K- 12 (Ref. 17). An accurate @I physical map ofthe B. s&i/b 168 gcnomc has rcccntiy been gencratcd by gene-dircctcd mutagenesis and the physical and gcnctical maps combined into a single structural/tiinctional map Ix. This has given an cstimated gcnomc size of4165 x 10”bp. Currently, eight European and six Japancsc laboratolics are collaborating to scqucncc the entire genomc in a programme expected to take about five years. As well as being deposited in the major DNA s~qucncc banks (Gcnl3mk and EMBL), scqucnce data Gotn B. s&i/i\.* is stored and analyscd at Trinity ColIcgc, Dublin, Ir&nd I‘),The Dublin database currently contains 410 protein-coding gcncs/ORFs (open reading frames), or about 10% oft& genomc.

249 reviews

The genetic analysis of B. srrbtilis has been greatly facilitated by the establishment of a centralized culture collection. The BncillrrsGenetic Stock Center”‘, cstablished in 1978 at Ohio State University, currently has a stock of _ 1200 strains, most of which are derivatives of B. srrbtilis 168. The Center maintains a strain &abase, and .:cgularly publishes an updated cataloguet. Cloning vectors ’ The dcvdopment of recombinant DNA (rDNA) techniques for B. sub&s is now well advanced. Pcrsistent problems with vector stability and with limitations in DNA transfonnation have been largely overcome. Staphylococcal plasmids, which formed the bases of the first generation of Bclcilltrs vectors”, arc still used for many applications. The B. wbfilis ‘cquivaicnt’ ofthe E. cdi vector pBR322 is plasmid pUl3110, a kanamycin-resistance plasmid isolated originally from Staphylococcus nc~ret&23. Plasmids such as pUBll0 had many limitations as cloning vectors (e.g. only a limited number of unique restriction sites for cloning, the lack of a suitable VEX fnr insertional inactivatlon and low transfonnability). A second gencration of ,RaciL piasmids involved the construction of chimeras between E. 4,li and staphylococcai plasmids. Tl.ese so-called ‘shuttle’ or bifunctional plasmids’+ enabled the initial cloning experiments to be carried out in E. oli, thereby exploiting this organism’s highly efficient transformation system. The use of shuttle plasmids also showed that, while E. co/i genes are not normally cxprcsscd in B. subtih, genes of 3. mbtilis are usually expressed in E. co/i. The failure of B. s&ilis to express the majority of genes ti-om Gram-negative bacteria appears tb relate to !imitations at both transcription and translation. Although E. ctdi and R. sttbtiks have virtually identical vcgctativc promoter consensus sequences (TTGACA 16-l 9 bp TAATAT), there is evidence that additional structural features are required for e&cicnt promoter utilization by B. srrbtilis RNA polymerase, particularly for an ATrich region upstream of the canonical -35 hexaurc+. Similarly, the majority of E. m/i ribosome binding sites (RBS) appear to function incfiricndy in B. ctr6Cli.i. Measurements OF the free energy of binding (AC) of the 3’ end of 16s ribosomal RNA (rRNA) to B. subtilis ribosome binding sites have shown that there is a higher degree of complcmentarity between the ribosome md RBS in B. s&tilis (average AC = -17.6 kcal mol-I (-73.92 kJ mol-I]), than ‘between those of E. npli (average AC = -11 .O kcal mol-1 [-43.72 kJ mol-‘j)‘“. Vectors based on staphylococcal plasmids, including shuttle vectors, show varying degrees of structural and segregational instability; a problem often made worse by the presence of cloned hetcrologous inserts. Studies, primarily in the laboratories of Bron and Ehrlich’7.‘8, have revealed the nature of much of this

instability, and have shown it to be a consequence of the phagc-like, single-stranded mode of replication of these plasmids. This can result in deletions, singlestranded intc> -1lediatcs and molecules of high molccular weight. in many casts, the extent to which thcsc aberrant corms are produced depends on the location and size of the insert DNA. More recently, both stable single-stranded replicating plasmids and @-replicating plasmids have been identified and are being dcvclopcd as a new generation of BnciNlfsvectors”‘. Certainly the former show exccptional stability, in batch and in continuous culture. Many of the technical tricks drvclopcd for E. m/i VCCKO~S have been adapted for B~cillrrsvectors, including the use of E. co/i IMZ ru-complcmcntation and other reporter genes, Ml3 polylinkcrs and phagc fl replication origin for subsequent production ofsinglcstranded DNA in a suitable E cdi host”. In addition. vectors developed by Youngm:‘? and hia coiicapcsf” permit transpositional mutn;enesis, transcriptionai fusion to la& d;x! LWZ -’ recovery of DNA adjacent to the site of integration of the transposon Tn9 17. The development ofphagc vectors for P. strbrilis has lagged well behind that of E. c&, and no phage vectors with the range and versatility of those of X exist for this organism. Gcnomc libiraries can bc generated and stored in E. co/i for subsequent transfer to B. sttblili+“, but this approach has limitations, particularly since a number of Bacillus gems appear to bc toxic when expressed at even mod-rate levels in LT. c~li. Alternatively, libraries can bc generated in d&vat&s of Bncilltu pbages such as $105J and SPP (Ref. 13). Attempts to dcvclop a cosmid system for B. s&&j have failed due to the lack ofan efficient irz rpi/ro packaging system for any of the candidate phagcs. Promoters Various special-purpose vectors have also been devclopcd to maximize the expression of native 2nd hctcrologous genes in B. dril:‘s, both intraccllularly and secreted into the growth medium”‘-3’. Initially, this expression was directed from promoters isolated from 5. srrhtilis and related spc-ties by use of promoter probe vcctorsIJ. These vectors contain promorerlcss reporter genes prececdcd by uniqac clcning site(s). When DNA fragments cloned at these sites contain appropriately orientated promoters, expression of the reporter gent is induced and this may be dctectcd by growth under appropriate srlcction or screening conditions. Examples of genes used as reporters in B. .&Ifilis are given in Table I _Such vectors have pcnnittcd the isolation of a number of efficient promoters, including a constitutive vegetative promoter from bacteriophage SPCtl (Ref. 34). Although the dcvelopmcnt of systems for highLevel, controlled expression of genes in Brlcillr~sis not as well advanced as it is in E. cd, promotcn that are constitutivcly expressed, as well as promoters whoso expression may be controlled by chemical or tempcrature induction, have been developed for B. srbti/LP+. TIBTECHJULY 1992 WLlO)

250

reviews Table 1. Repotter genes used in Bacih~s su&ih Reporter gene

Enzyme

Method of detection

amyF

ar-Amylase

Zones of clearing around colonies on starch plates 4myfase assay

cat86

Chloramphenicol acetyltra!i;ferase

Chioramphenicol resistance Chloramphenicol acetyl transferase assay

!acZ

p-Galactosidase

Blue colonies on X-Gal platesa Fluorescent colonies when soraved with MUGb @Galaciosidase assay

llJxAf3

Bacterial luciferase

Scintillation counter Luminometer Photographic film

xylE

Catechol 2,3dioxygenase

Yellow colonies when sprayed with catechol Catechol2,3dbxygenase assay

aX*G;! = 5_brom&cMorc-3-indoCp_gafactoside

IMUG = 4-methylumbellifer$-p-o-galactopyranoside These include promoters from well-characterized Baths germ such as the stationary phase-induced or-amylase gene from B. alllyloliq1r~~fo~‘~?rs35 and the sucrose-inducible sncR control region of B. srrbdis levansucrase”“. Another potentially us&l promoter is the thermo-inducible promoter system based on the temperature-sensitive immunity protein irom Bocillrrs phage 4105 (Ref. 37). In addi’:ion to promoters isolated from Grampositive bxctcria and their phages, a series ofpromoters has been developed for use in Bxih strains that are either i:!:irely of Gram-negative origin or which contain elements of both Gram-positive and Gramnegative origin. Such promoters have been used for high-lcvcl, controlled synthesis of heterologous proteins and include the hybrid P,l,,o promoter system, consisting of phage T5 promoter PNzs and the E. co/i iac operator33JR, and the hybrid spa-1 promoter system, consisting ofa phage SPOl promoter 2nd the E. coli lacoperator”. Both of these promoters arc induced by isopropylthiogalactopyranoside (IPTG). B. subtilit can be used for intracellular production of hcterologous proteins, but only in specific cases is this organism a more amenable host than E. CC&.The main advantage of B. srtbfili~and other Bncillifor hcterologous protein production lies in their ability to secrete proteins directly into the growth mc&m. This is facilitated by the use ofsecrction vectors, a number ofwhich have been described and use,d with a variety of heterobgous proteins and with rather mixed successJQ0.

Although natural transformation is highly efficient in taking up lineat fragments of homologous DNA, it is very inefficient for the uptake of plasmid DNA.

______-___ TI~~TECH JULY 1992(VOL IDI

Only multimeric plasmid DNA or monomers containing internal repeats are transformed efflcientiy’9. Monomeric plasmid DNA can, however, be ‘rescued’ by homologous recombination with a re!ated resident plasmid The limimtions of natural transformation have led to the development of other transformation prois cedures for B. srtbtilir. Ptotoplast transformation relatively efficient, with up to 10% of the regenerated protoplasts containing the plasmid DNA under saturating conditions”. The technique is effective for monomers of plasmid DNA and has been adapted for 2 variety of Bwillrrs species including R. nmylo/iqkc&rciens, B. nwthr&, 13. Wretri&tzii and B. sr~~7rodlerrilophihrs”. The main disadvantages are (1) the laborious nature and lack of reproducibility of the methods, (2) the need to use complex media that precludes certain positive selection procedures, and (3) the relatively long time (36-4X h) required for regeneration. More recently, electroporation techniques have been developed for B. subrilir which, while not as ef ficient as those developed for E. cdi, nevertheless give between 103 and 1Oj transformants pet microgram of plasmid DNA”‘. Elcctroporation methods have also been developed for other species of Bncilhrs including B. arnylolipe@ciens, 3. ceretrs and B. tkkr@e~~siP. Bacillus and biotechnology

iBririZ!us strains are widely used as important sources of industrinl enzymes, fine biochcmicals, antibiotic: and insecticides. Their ability to secrete large yuantities (grams per litre) of protein directly into the growth medium. the ease with which they can be grown and their well-proven safety, has also made them prime candidates for the production of heterologous proteins.

The world sales for industriai enzf_mes (amounting to 75000 tons) was recently valued at US$300-600 x 1O”pcr annum. About 75%, by weight, of these are hydrolytic enzymes (two-thirds of which are proteolytic enzymes used in the detergents, dairy and leather industries and one third comprises the carbohydrases used in the baking, biewing, distilling, starch and textile industries)tJ. Although most species of E&ci!lcrrsecrete proteases and cx-amylases, the properties of these enzymes and their activities vary 6om strain to strain. The main classes of enzyme and their producer si;_ains arc !isted in Table 2. Mauy of these cuzymes have been cloned and subsequently expressed in B. s&/is and B. Uenij&~b, in some cases after modification of their propertics by protein engineering”. B. srrbtilis produces 2 wide variety of extr2cellular enzymes, aithough only a few are produced on a commercial scale from this organism (Box 1). BuciHl4sspecies have also been used to produce a number of intracellular enzymes on aq industrial scztlc, including glucose isomerase-‘“, thermostable enzymes such as glucokinasesJfi, and a variety of restriction

251

veviews endonuclcases such as Barr1H1, Bdi, B$, Bg&I and B4 (Ref. 47). Most commercially important Brtcil!rrs strains are easily grown and maintained in submerged culture at high growth rate using a range of carbon and nitrogen sources. Knowledge of their genetics, biochemistry and fermentation ~~opcrties has led to extensive strain-development programmes which, together with improvements in culture media, have resulted in strains with better growth, product yield and secretion characteristicsJ7. These may have been achieved by a combination of traditional mutation-and-selection regime+ and, more recently, by cloning the genes for the products conccrnedq?. However, because of the commercial interest, ~116 results of industrial straindevelopment programm!zs are rarely published.

The cfficicntlarge-scale production ofheterologous enzymes and proteins by B. dtilis has been the subject of considerable res-arch effort in industrial and in academic laboratories. The ability of this organism to secrete proteins directly into the culture medium, rather than accumulating them as insoluble aggregates (inclusion bodies, IBs) intracellularly or in the p&plasm, offers considerable process advantage@. Afier the removal of the producer cells, the protein products can be purified either by conventional technology (e.g. size exclusion or ion exchange chromatography) or by the newer aflinity tagging tcchniquess’. Other species of Bdhts are likely to prove useful for development as hosts for hetcrologous protein production, particularly for producing low levels of extracellular protcascs. Of particular note here are

Table 2. Major industrial enzymes produced by Llaciffusspp. Enzyme

Producer strain(s)

wAmylase

B. amyloliquefaciens 6. circulans 6. licheniformis B. stearothermophbs 5. subtilis

P-Amylase

B. polymyxa

Alkaline phosphatase

B. licheniformis

Alkaline protease

5. arnyloliquefaciens 5. IichenMnis B. subtilis

Cyclodextran glucanotransferase

B. macerans 5. megatenum

pGlucanase

5. subtifis

Glucose isomerase

B. coagulan s

p-Lactamase

6. licheniformis

Neutral protease

5. amyloliquefaciens

Pullulanase

Bacillus spp.

I

Box 1. Major extracellular enzymes of B. subtih a-Amylase, Alkaline phor, .dtase, Alkaline protease, Aminopeptidase, Arabinase, Cefiulase, Dextranase, DNase, endc-Nacetylglucosaminidase, exo-Wacetylglucos:Sninidase, exe-Nacetylmuramidase, Estara% Galactanase, (3_1,3Wcanase, Levansucrase, Maltase, Nacetylmuramyk-alanine amidase, Neutral proiease, 3’-nucleotidase, 5’-nucleotidase, Pectate lyase, Xylanase. Taken from Ref. 43.

species, such as B. brevic, that produce su&ce or s-layer proteins which foml a crystalline barrier at the outer surface of the ccl1 wall. These proteins have been observed at concentrations of up to 12 g l-1 in the cultilrc medium and, consequently, attempts have been made to incorporate the expression and signa! sequences of their gcncs into secretion vector9. In common with other Frokaryotes, B. nrbrilic is not able to glycosylate proteilrs during export, and conscqucntly cannot bc used to produce fully authentic mammalian proteins. However, for pharmaceutical proteins in which biological activity, rather than total structural authenticity, is the more important factor (e.g. for diagnostic tests, the chemical modlrication of drugs or vaccines), B. nrbfilis could rcprcscnt n costeffective producer organism with particular advant::gc; over E. co/i.

The ccl1 envelope of B. s&i/b is simpler than that of Z 4, since it has no outer membrane, associated lipopolysaccharidcs (LPS) or a mcmbranc-enclosed periplasmj3. The absence of lipid A moieties of LPS reduces the potential for pathophysiological effects (e.g. fever, endotoxic shock, hypotenzion) from copurified endotosins, and the absence @fan outer membrane simplifies the secretion process since rsop:oteins need only be translocated through a single membrane (and the cell wall; see below) before reaching the culturc medium. In contrast. in E. co/i the main criport pathway &cc% p:otcins iLit either die peripiasm or the outer membrane. Production systems in E. co/i therefore usually involve directing the protein into the periplasm from which they are released by osmotic shock. Even in the periplasm, high levels of production can still lead to the formation of aggregates in E. co/i, with attendant problems of disaggregation and rcnaturation into authentic or biologically actiw stmctures.

Despite i:j apparent advantages, the application of B. srrh:iiis to the production of hetcrologous proteins has been limited. A major pi-oblem has been the production of at least seven extracellular protcases which cause substantial degradation of secreted foreign ~TOteinP. These include: neutral protease A, Subtilisin (alkaline protcase); a minor extracellular protrasc: a mctalloprotease; Bacillopeptidase F; neutral protcasc B; and a minor serine protease. Strains lacking all but one of these enzymes have been constructed, reducing the level ofprotease activity in the culture meditiirl

----

--__

TIBTECH JULY 1992 !VOL 101

r_1

Components of bacterial protein= secretion pathways

/ Se&

I

Cytosolic components

Membrane peripheral components (internal)

GroEL DnaK

GroES GroEL DnaK

SecA

SecA

SecE

/

SecY

SecY

1

I iep

SPasel

SecD

Membrane periphe?al compOnSntS

Extrscellularcomponents

(SXtSrnSl)

SecF

PrsA Fe(M)

Figure 2 Schematic representation of the components involved in the Sec.dependent translocation of proteins across the bacterial cytoplasmic ment brane (redrawn from Ref. 78). Exoproteins, synthesized on ribosomes as preproteins (mature protein with N-terminal signal-peptide exten sion). interact with chaperonelike protein SecB which prevents their folding into secretion-incompetent forms. SecA, a peripheral mem brane protein associated with the SecY-E-l translocase complex, interacts with the SecEpreprotein complex via affin’hyfor Sect3 and both signal and mature domains on ihe preprotein. SecA generates energy for translocation by the hydrolyses ATP. Once trznslocated through the membrane via the SecY-E-l complex, the sign;: pcptide of the prepiotein is cleaved by a signal peptidase to release the mature protein. Many Bacillus exoproteins are synthesized with an addiiional N-terminal sequence, the ‘pro-’sequence, between the signal and mature domains. The pro-sequence, which is oroteolytically cleaved subsequent to translocation, may also function to maintain a secretion-competent form throughout the secretion process. The inset lists the components of the protein secretion pathways in E. &and in B. subtifis fhomo!ogues are shown on the same line); 1 represents a polypeptide of unknown function that co-purifies with SecE and SecY subunits; Fdlll) appears to perform a chaperone-like function required for a late translocahon step and extracellular folding of levansucrose.

to less than 0.5% of that of the wild type. Published data indicate that heterologous proteins produced by protease-deficient strains usually show significantly improved stabilitys+. TlbcCHJULYI992 NO1 10)

A second major restriction on the use of B. s&i/is has been the limited understanding of its secretion pathway, particularly of those componcntskagcs that arc unique to this organism. In recent years there have

253 reviews

been significant advances in our understanding of the Other products molecuiar mechanisms of Bacill~rssccretionss. HomFine biochemicals ologues for some of the E. culi sccrction pathwav In addition to the production of industrial enzymes, components (Fig. 2) have been identified in B. s&i/& B. strbfiiis has been used cxtcnsivcly for the produc(Fig. 2, inset)““-“‘. Notwithstanding the basic simition of valuable primary mctaboiites, such as hypolar?;:.: with their Gram-negative counterparts, the B. xanthinc, inosinc and bmanosinc, for chemical phossrtbfilic export components are suf&icntly diffcrcnt to phorylation to xanthanylic acid (XMP), inosinic acid merit a thorough analysis in their own right. In ad(IMP) and guanylic acid (GMP), which arc of comdition, the implications ofhaving no outer membrane mcrcial importance as flavour enhanccrsfis. The nuclcoand a markedly different ccli wall, together with the sides arc the primary fermentation prodilct because identification ofproteins (e.g. PnA)“” for which there their greater membrane pcrm>ability allows them to are no E.co/i homologues, suggest that the study of the accumulate to much higher concentrations (than the secretory process in 8. arbtilis will reveal novel features. nuclcotides) in the culture medium. proteins exiting on the cxtcmal surface ofthc B. srrbExtensive knowledge ofthc biochemistry and rrgutilis cytoplasm&z membrane cncountcr the ccl1 wall lation of B. s~rbfil~~ biosynthetic pathways has alltiwcd bcforc they arc free to enter the culture medium. The growth and rational mutant-isolation stratcgics to bc wall thercforc represents the iinal stage of export. The dcvelopcd to the extent where this organism has been B. srt6tilis cell wall is a highly crosslinked semi-porous considered for the production of a range of other structure, composed primarily of peptidoglycan metabolitcs at commercially viable Icvcls~~. Attempts (-40%) and anionic polymers. Its composition and have been made t9 dcvclop B. s&i/is stram K for the structure varies with culture conditions (c.g. pH. ionic production of amino acids such as Trp, His and l’hc, strength, osmolarity, ccl? density and nutrient supply) although not yet to production levels that compctc and consequently its porosity and ion-exchange ca- with those of Brcrribncrerirrrn, Cov),lrc~ncleril,rir or Sfrrofia spccics. pacity are also variable hl. Although some exoproteins may be able to diffuse freely through the cell wall, the passage of larger proteins, or proteins that arc folded Antibiotics in such a way as to cxp~sc extensive positivcIy charged Bociil~rsspecies produce a variety of antibiotics6’J residues at their su&ce, arc likely to be affcctcd by including: (1) cyclic oligopeptidcs, such as bacitracin, the wall’s molecular-sieving and/or ion-exchanging that inhibit cell wall synthesis; (2) mcmbranc-active properties. Whilst homologous cxoproteins are comlinear or cyclic oligopeptidcs such as gramicidins and patible with export through the B. ar6rili.c ccl1 wall, polymyxin; (3) basic pcptides, such as edcines; and (4) other proteins may be retained by the wall, possibly as aminoglycosidcs that intcrferc with ribosomc function. Although B. .~rbrilis strains are not prodigious proteasc-sensitive, partially folded structures. The abi?ty to secrete human serum albumin and atria1 producers ofantibiotics, scvcral bioactivc compounds have been found (e.g. bacilyain and subtilin) in repnatriuretic a-factor from B. strbtilir protoplasts, but not of the rcscntativc strains. Strains of B. dtilis also product a from whole cells, suggests an involvement wall”?*““.Many other proteilis have been found to bc biodegradable surfactant (sulfactin), a cyclic lipopzppoorly secreted from B. subrilis, including lysozymc, tide compound with exceptional biosurfactant activE. coli outer membrane protein A (OmpA) and cerity (set A. Ficclltcr, TlBTECHlO, 208-217, 1992). tain cy-interfcrons, although it is not clear at what stage their secretion is blocked’“. Insecticide? Despite thcsc problems, high levels ofsccretion have Although many bacteria cause diseases of insects, been obtained with a number of hcterologous proonly a few have been used commercially as a means teins. These include human a-intcrfcron (15 mg 1-l). growth hormone (200 mg 1-l) and epidermal growth Table 3. Examples of insecticidaltoxins produced by factor (EGF) (240 mg I-‘), porcine pcpsinogcn (>5GO Bacillus spp. mg l-t), pcrtusS.s toxin subunits (2-100 rng I-') and Gram-negative periplasmic protcins3s@. Signiticantly, the levels of production in thcsc examples are not comparable with those obtained for homologous Honey bee larvae 6. larvae Scarabaeid beetle proteins such as a-amylase. B. lentimorbus larvae Unexpectedly, B.shillhas also proved useful for Scarabaeid beetle 5. popilfiae the high-level intracellular production ofoutcr mcmlarvae brane proteins from vimlent Gram-negative bacteria”‘. Lepidopteran larvae B. sphaericus These proteins, which have potential as vaccines and Lepidopteran larvae 13.thuringiensis sibsp. aiwawai for inimunodiab~ostics, can be produced in B. s~tbtilis Soodopteran larvae l3. thuringiensis subsp. entomocidus free fPom the endotoxins with which they nomlally Dipteran larvae 6. thuringielsis subsp. israeJensis Lepidopteran and foml tight associations when produced in their natu13. thuringiensis subsp. kurstaki dipteran larvae ral host. Other proteins that have been produced mtraColeopteran larvae B. thuringiensis subsp. san diego cellularly are pertussis toxin subbunits and tissue plasLepidopteran larvae B. thuringiensis subsp. thuringiensis minogen activator (t-PA)‘6Jt7.

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SPOOF-P

SPOOF

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Figure 3 The phosphorelay signal-transduction pathway for the induction of sporulation in Bacillus subtilis. Multiple cell and environmental signals are sensed at various points throughout the relay and may function positively or negatively in relation to the induction of sporulation. The response regulator, SpoOA, attaches to specific nucleotide sequences iOA boxes) at or near the promoter0 of genes involved in the control of ‘sporulationor post exponential growth phase responses to activate or repress their activity. SpoOA-P negatively regulates abrfl, the product of which is a repressor of transition-state genes such as chemotaxis and competence, and positively regulates a number of sporulation genes, includicg spollA(which encodes U% Additional phosphorylated intermediates are found between the histidine protein kinase (HPK) sensor components KinA and KinB, and SpoOA-P. These intermediates permit the additional signal inputs that are required to process the complex range of cell and environmental messages before committingthe cell to sporulation. (Redrawn from Ref. 76.)

val stage of lepidopteran, dipteran or coleoptemn insects, the pro-toxin passes unchanged through the acidic stomach, but is proteolycicaliy cleaved into the active form in the alkaline mid-gut. The activated toxin paralyses the gut and mouth parts and alters the gut permeability to ions such as K+. The toxin binds to specific recepcoTs on the surfscc of the gut, a fcature of their mode of action that confers their commercially valuable specificity. A number of toxin (cry) gents have now been sequenced and grouped into classes that reflect both their protein structure and their activity spectra. Sitedirected mutagenesis is permitting analysis of the receptor sites in the toxin molecule and its mode of action. In cases whcrc a toxin has a cpmmcrcially important specificity, but has a low level of toxicity, the design of hybrid toxins can now bc considcrcd. More recently, toxins active against flatworms, mitts and protozoa have been discovercd7’. TIlc USC of BnciNrrsinsecticidal toxim represent an clcgant and environmentally benibm prospective solution to the increasing problems of resistance to chemical insecticides, and to the persistence of these compounds in the environment. To assist their application to crops, cry genes have been cloned and expressed at high !cvcls in Psefr~~~rttoizosf7rf~)res~ei1~. The Pscurlonrorrns hosts are killed by chemical fixation to prevent spread beyond the site of application, but the, toxin is not destroyed hy the treatment and is active for some cimc. cvy gcncs have also been cloned and cxprcssed in susceptible plant species, and in a bacterium that colonizes the xylem of piants, thus providing systemic immu. lity against insect pests”. The current tnarkct for B&/m insecticidal toxins is still relacivcly small by comparison with chemical insccticidcs (Cl’%), but is growing at an estimated 1 1% per annum. It is to be hoped that their lack of general toxicity and high degree of specificity to their tagct organism.;, combined with their lack of persistence in the environment, will eventually outweigh their significant cost disadvantage in an increasingly environmentally conscious world.

Sport&ion The existence ofcndospores was first obscrvcd in B. srrb&P, and simultaneously, in B. ar1t8rncis’~over 400 ycan ago. Endosporcs have subsequently been the ofcontrol. Insecticidal toxins are produced by a numsubject of intense investigation, that has led to dcfiber of Badlllrs species, in&ding B. f!~rri@~sis (see nition of the physical properties af spores, their biochanges J. Van Rie, TIBTECH 9, 177-179, 1991; and K. J. chemical composition;, the morphological Brunke and R. L. Meeusen, TIBTECH 9,197~200, accompanying sporulation and their gcnctics. Sporu1991), B. lowtie, B. Irrrfirrlorblrs, B. p@ililz and B. I,ation in B. srrbrilis now constitutes the best-studied splrrlericrrs, and have potential for use as commercial example of a prokaryotic differentiation proccs+. insrcticidec against insect pests”’ (Table 3). In recent years, the emphasis has been on undcrThe insecticidal toxin is produced during sporustanding ch:: control of the sequential cvcncs that lead lation as an inactive pro-farm of between 27 and 140 to spore formation. This work is ofapplied importance kDa, and can account for 30% of the total dry weight since many ofthe products produced commercially in of the spore. These toxins are much more potent than Batilirrr (including insecticides, hydrolases and antiother insecticides, being 300 times more potent than biotics) are produced concomitantly with sporulation’. synthetic pyrethroids and 80000 times more potent New concepts in the control of differentiation prothan organophosphates7’. When ingested by the lar- cesses have been dcvclopcd horn the study of Bacilhts

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that are applicable to other organisms (e.g. Streptont~ces)~~that are also of commercial importance. This work has shown how alternative, or minor, o-factors can interact with core RNA polymcrase to change its specificity for different classes ofpromotc&. To date, nine cr-factors have been identified in C. s&&s. In addition to the vcgccativc n-factor (I+) (43 kDa in B. s&/is as compared with 70 kDa in E. cnli), at lcast four (GE, crF, & and a”) are specific for sporulation genes, three (19, 5” and aH) function in stationary phase, and one (uL) during nitrogen starvation. In addition to progress on the control of sporulation, considerable interest has been shown in events leading to the induction of sporulation and other events that occur towards the end of exponential growth. Initiation of sporulation rcquircs the intcgration ofboth external and internal stimuli including the nutrient status of the cell, cell density and cell cycle signals7G. Sporulation is induced through a phosphorelay that is a variant of a two-component stimuIus-response mechanism” (Fig. 3). demonstrates clearly The B. ruhtilk phosphorclay how multiple, sometimes contradictory stimuli can be processed to elicit a specific response. The expression ofmultiple virulence gents by a number ofpathogenic microorganisms (e.g. Bwdetelko p~rtrrssic, Stopkylococctrs amus and Vihrio chulerac) also appear to be controlled by variations of the two-component pathway. However, neithCr the nature of the environmental signals involved, nor the details of the signal transduction pathways have been fully c!ucidated. The R. srrbM_r phosphorelay, control& sporulation in 2 nonpathogenic micoorganism9 is therefore providing valuable insights into the mechanisms by which bacteria sense and respond to multifactorial changes within their environment - these are likely to have a significant impact on clinical microbiolcg, and also for an increased role for B. s&i/is in industrial microbiology.

(Hanvood, C. R. and Cutting. S. M., cd*), pp. 221-266,john Wiley 16 P&x, I’. J., Amjad, M., Wu, I-I., Sandoval,I+. and Carrro. t. (IVSO~

References

9 Hoch. J. A. (1991) McrlindsE~~z:rvtal. 204,305-320 10 Dedondcr, R. A., Lepesant,J-A., Leprsanr-Kejzlarova, J., BIltautt.A., ~Clin~i~iol. 33, Sreinmetz. M. and Kunst, F. (:977j Appl.Ettninw~ 989-993

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The miwobialproductionof epoxides David J. Leak, Peter J. Aikens and Mahmoud Seyed-Mahmoudian Chiral compounds in which biological activity resides in only one of the enantiomers are, increasingly, being demanded in enantiomerically pure form. The use of biocatalysts for production of epoxides as chiral intermediates in the synthesis of these compounds has realistic commercial prospects, but also presents formidable technical problems.

nrisiug from the elcctroncgativity ofthc osygcn), they possess considerably higher reactivity than that norui:~lly associntcd with cthcrs. Thus, cpoddcs rc3ct rcedily with a iargc number of rcagcnts, including n~c!rc~phil~~r. c!cctropli3lcs, acids, bases. reducing agents nnd Sony oxidizing agents (Fig. 1). Nucleophilic ring-opening with osygen, suiphur, nitrogen or carbon nuclcophilcs is the most-commonly used proccss, with attack gcncrally occunicg at the stcricnlly Icss-hindcrcd carbon, resulting in inversion of stcrcochemistry at this position (Fig. 1). In nonterminal cpoxidcs (SW Glossnry) this nlcans that the TIBTECtt JULY t992-(VOL 10)

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1992, Elsetier Science Publishers Ltd (UK)

Bacillus subtilis and its relatives: molecular biological and industrial workhorses.

The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of ma...
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