Activity of Mutant o-F Proteins Truncated near the C Terminus

Vol. 174, No. 22 JOURNAL OFBACTERIOLOGY,Nov. 1992,p.7144-7148

0021-9193/92/227144-05$02.00/0

Copyright

©

1992,AmericanSocietyfor Microbiology

Activity of Mutant o-F Proteins Truncated near the C Terminus

K.-T. MINANDM. D. YUDKIN*

Microbiology

Unit, Department

of Biochenustry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

Received 25 June1992/Accepted16 September1992

(F',

the product of the

spoILAC

gene of Bacilus

subtilis,

is

homologous

in amino acidsequence throughout most of its length with several othersigma

factors of

B.

subtUis

and Escherichiacoli.

However,

8 residues from theC terminus the homology

abruptly

breaks

down, suggesting

that the C-terminal tail of theprotein may be dispensable. It is known that an amber mutation at the 11th codon

(wild-type glutamine 245)

from the C terminus abolishes thefunction of the sigma factor. We have now

placed chain-terminating

codonsatthe ninth codon

(wild-type lysine 247),

the

eighth

codon

(wild-type

valine

248),

orthe seventh codon

(wild-type glutamine

249) from theC terminus.

We

have

tested

the

resulting

mutants for their

capacity

to

sponrlate

and for their

ability

totranscribe from a promoter

(spoIHIG)

that isnormallyreadby RNA

polymerase

bound tocf

(Eu").

The

results

indicate that a mutant

o' lacking

theterminal 7 residues functions almost

normally,

which

suggests

thatglutamine 249 is

dispensable.

By contrast,

lysine

247 is crucial for the

activity

of

cF:

deletion ofthe 9

C-terminal

residues

totally

inactivates the

protein.

When theterminal 8 residueswere

deleted, placing lysine

247at the

C terminus,

the

transcriptional activity

of thefactor is reduced by about 80%o: we attribute this effect toneutralization of thepositive charge of

lysine

247by formation of a salt

bridge

with the

-COO

terminus.

When

placed under starvation conditions, cells ofBacillus subtilis initiate

a

developmental

process

that leads, after several hours,

to the

formation of heat-resistant endospores.

Early in development, an

asymmetric

septum

divides

the

sporulating

cell

into

two compartments, the

forespore

and

the mother cell, in which

gene

expression

occurs

differen- tially (15).

As

sporulation proceeds, the cells manufacture

a

succession

of

sigma factors, each

of

which directs

the RNA

polymerase

to

transcribe certain sporulation-specific

genes

(6, 19, 31).

Because

these sigma

factorsarenot

needed for growth, they

can

be altered

without

affecting

the

viability of

the

cell; they

are therefore

ideally suited

to

studies

of structure and

function. Moreover,

because

much of the amino acid

sequence

(and therefore presumably the

struc-

ture) is conserved

between

different sigma factors, such studies of sporulation-specific

factors may

also provide

valuable

information about

the

major sigma factors that

direct

transcription during vegetative growth of

B.

subtilis and other bacteria (10, 12, 14).

In

the known sigma factors, much

of

the C-terminal one-fifth of the

sequence

is strongly

conserved. Thispart of the

protein

contains aregion gener-

ally believed

to

constitute

a

helix-turn-helix motif,

which

interacts

with

the

-35

region

of the

cognate

promoters

(2,

9,

29), followed by

a

short region rich

in

basic

residues. After

this basic region, conservation of the

sequence

abruptly

breaks

down,

so

that the C-terminal tails of the proteins have

noapparentsimilarity

(Fig. 1A).

The

experiments described

in

this

paper are

concerned with the C-terminal region of orF,

one

of the sporulation- specific sigma

factors of B.

subtilis,

and were

designed

to

show which residues

of the basic

region

and variable tail of this

protein

areessential for normal function. We know from earlier workthat a mutation that removes the 11 C-terminal residues

(3

residues from the conserved basic region plus the 8 variable residues of the tail) abolishes function (34). We nowdescribethe effect of mutations that remove the 7, 8, or 9 C-terminal residues.

*

Corresponding

author.

MATERIALS

ANDMETHODS

General methods.

Chromosomal

DNA from B.

subtilis

was

purified

as

described by Ward and Zahler (33).

B.

subtilis

cells were

made

competent for

transformation by

the

method of Anagnostopoulos

and

Spizizen (1).

Transformants were

selected

on

Oxoid nutrient

agar

containing chloramphenicol (5 ,g/ml)

or

erythromycin (1 ,ug/ml) and lincomycin (25

Ag/ml) ^and

also

containing 0.02% 5-bromo-4-chloro-3-in- dolyl-j-galactoside.

DNA

and Escherichia coli manipula- tions

were

carried

out

by standard methods.

Geneticconstructions. The

strains and plasmids

used are

listed

in

Table

1.

Plasmid pTl

was

constructed

as

follows.

A

0.87-kb frag-

ment

containing the spoILAC

gene was

obtained by digesting plasmid pSG-EP with ClaI, filling in, and digesting it again with HindIII. This fragment

was

ligated

to

SmaI-HindIll- digested pBluescriptlIKS. The

cat gene

from plasmid pSGMU2

was

ligated into the ClaI site

of

the resulting plasmid

to

yield pTl.

To

integrate

the cat gene into the chromosome between

spoILA

and

spoVA,

we

digested pTl with SalI, filled in, and ligated

it with the

2.2-kb fragment

containing part

of the spoVA

gene

obtained by digesting pSGMU187

with

PstI

and

filling.

We

transformed the ligation mixture into strain 2002 and selected for chloramphenicol resistance. Chromosomal DNA

from the resulting strain

(K1)

was transformed into

strain spoIIAC1

to

make M-11;

this strain is

chlorampheni- col resistant and contains spoIIACQ245(Am),

a mutation

that lies close

to the

3'

end of the

spoILUC

gene. We introduced a mutation into the cat gene of

pTl

by

restricting and filling the

NcoI

site and religating. The resulting spoIIUC+cat plasmid

was linearized with SacI and used to

transform

M-11to

sporulation proficiency

andchloramphen- icol

sensitivity. Spo+

colonies were selected on Schaeffer's

sporulation

agar

(27)

after chloroform treatment

(11). This series

of steps

generated

strain K3.

We

introduced mutation spoIIACQ249(0c) into plasmid

pTl by using

the

Bio-Rad

mutagenesis kit based on the method of Kunkel et al.

(13).

The

oligonucleotide

5'- 7144

VoF PROTEINS TRUNCATED NEAR THE C TERMINUS 7145

total number of residues I HTHmotif ^f basic -j

70

a--- TLEEVGKQFDVTRERIRQIEAKALRKLRHPSRSEVLRSFLDD 32

--- TLQELADRYGVSAERVRQLEKNAMKKLRAAIEA

A--- TLEEVGKVFGVTRERIRQIEAKALRKLRHPSRSKRLKDFLE

--- SQKETGDILGISQMHVSRLQRKAVKKLREALIEDPSMELM C --- TLTEIGQVLNLSTSRISQIHSKALFKLKNLLEKVIQ

--- TQKDVADMMGISQSYISRLEKRIIKRLRKEFNKMV

--- TQSEVAERLGISQVQVSRLEKKILKQIKVQMDHTDG

- --- TQMEVAEEIGISQAQVSRLEKAAIKQMNKNIHQ

e --- SYQEISDELNRHVKSIDNALQRVKRKLEKYLEIREISL B)

ae

---TQSEVAERLGISQVQVSRLEKKILKQIKVQMDHTDG M-7 ---TQSEVAERLGISQVQVSRLEKKILKQIKV

M-8 ---TQSEVAERLGISQVQVSRLEKKILKQIK M-9 ---TQSEVAERLGISQVQVSRLEKKILKQI

613 284 371 262 254 239 255 260 218

255 248 247 246

M-11---TQSEVAERLGISQVQVSRLEKKILK 244

FIG. 1. (A) Alignment ofbacterial sigma factorsatthe C terminus.Boldfacetypeindicates conserved amino acid residues (10, 14). (B) The Cterminiof themutant

e'

proteins.

TGATCCAT1TAAACCTTGATC-3'

was used to convert theglutaminecodonCAAatresidue249(whichis partof the spoILAC sequence carried on pTl) toTAA(Fig. 2A). This mutant plasmid ^was linearized with ScaI andwas used to transformK3 withselection forchloramphenicol resistance (Fig. 2B). In asimilarway weused themutagenic oligonu- cleotides

5'-TGATCCATTJTACYTTGATCTGT-3'

and 5'-TT TGAACCTAGATCTGT11-3' tointroduce mutationsspoII ACV248(0c)andspoIACK247(Am) (Fig. 2A). The changed

sequences ofthe mutantplasmidswere confirmed byDNA sequencing (26),with the syntheticoligonucleotide 5'-TAG GCCTAATGACTGGC-3' used as the sequencing primer.

The presence of the mutations in the chromosome was

confirmedbyDNAsequencingofpolymerasechain reaction (PCR)-amplified chromosomal products prepared with the two primers 5'-TTCGATCGGACGTGACGTC-3' and 5'- TGGTCTGTCGTACAGCGGTT-3'. PCR was carried out with the Perkin-ElmerCetusGeneAmp PCRreagentkit(24).

PCRfragmentswerecloned intopTZ19R and sequencedby usingtheprimer

5'-GTCCTTTGTCGATACTG-3'

toconfirm thatonlythedesired mutationwaspresent.

spoIIIG-lacZwasintroducedby transformingstrainswith DNApreparedfromstrain687,with selection for resistance toerythromycinand lincomycin.

Resuspension experiments. Strains of B. subtiliswere re-

suspendedinthe starvation medium ofSterlini and Mandel- stam(30).Times afterresuspension (in hours)aredenotedt1 and t2, etc. Samples (0.75 ml) ofculture were assayed for alkaline phosphatase activity and the incidence ofsporula- tionwasmeasured asdescribed by Erringtonand Mandel- stam (7). The substrate o-nitrophenyl-3-D-galactoside was

used to measure the

0-galactosidase

activity of culture samples (0.5 ml)(16, 18).

Immunoblotting experiment. Afterresuspension, samples (2 mlofculture)^weretaken att1 andt4. Polyclonal anti-&F antiserumwas used forWesternblotting(immunoblotting), whichwasbased on themethod described bySambrooket al. (25).Thecloning andoverexpressionofspoIUC, and the purification of anditsuseinimmunizing rabbits,willbe described elsewhere.

RESULTS

Toconstructstrains thatsynthesizetruncated(r proteins (Fig. iB),wewanted to introducenonsense mutationsinto the seventh, eighth, or ninth codon from the 3' end of spoIlAC, the structural gene for ai. We used in vitro mutagenesis to place the desired mutations on a plasmid (pTl) that contained the cat and spoILUC genes and then introducedeach mutationonto the chromosome byhomol-

ogousrecombination witharecipientstrain(K3)which had

amutantcatgeneimmediatelydownstream ofspoILAC (Fig.

2B).

Phenotpes andregulationofspoIIIG expression.The three resultingstrains whose construction is described aboveare

isogenic, differing only in whether

eF

^lacks 7, 8, or 9 C-terminal residues. (Forconveniencewe call these strains M-7, M-8,andM-9.) Theyareisogenicalsowith strainM-11, which lacks ¹¹ C-terminal residues. When resuspended in sporulation medium, strains M-7 and M-8 made heat-resis- tant spores at an incidence thatwas indistinguishable from thatof the wildtype(50to80%),whilestrains M-9 andM-11

provedtobe asporogenous(<10-7).

In wild-type cells,

er

is known to direct the RNApoly-

merase to the promoter of the sporulation-specific gene

spoIIIG (32).Toseewhether the mutantsigmafactors could

A)

VOL. 174, 1992

7146 MIN AND YUDKIN

TABLE 1. Bacterial strains and plasmids

Strain orplasmid Genotype orphenotype reference B.subtilis

2002 his-i

lys-l

ura-l 35

spoILAC1 lys-1ura-lspoIL4CQ245(Am) 4

687 trpC2fl(spoIIIG-lacZ erm) 23

Kl his-i

lys-1

ura-1cat' Thisstudy

M-11

lys-1

ura-lcat'spoILACQ245 Thisstudy (Am)

K3 lys-I ura-1 cat-I Thisstudy

M-7 lys-1ura-1 cat'spoIL4CQ249 Thisstudy (Oc)

M-8 lys-lura-1 cat' spoILACV248 Thisstudy (Oc)

M-9

lys-l

ura-l cat'spoIL4CK247 Thisstudy (Am)

M-7GL fl(spoIIIG-lacZerm)lys-I Thisstudy ura-1 cat'spoIIUCQ249

(Oc)

M-8GL fl(spoIIIG-lacZerm)lys-I Thisstudy ura-1cat'spoIIUCV248

(Oc)

M-9GL fQ(spoIIIG-lacZ erm) lys-1 Thisstudy ura-1cat' spoIIUCK247

(Am)

M-11GL fl(spoIIIG-lacZ erm)

lys-I

Thisstudy ura-1cat' spoIIUCQ245

(Am) E.coli

XLlblue supE44hsdR17 recA1

endA1

3 gyrA46thireUl1 F'(proAB+

lacPl lacZAM15TnlO(Tetr)

CJ236 dut-1ung-1thi-1reL41 13

pCJ1O5(Cam'F') Plasmids

pBluescriptIIKS blafl-or 28

pTZ19U bla fl-on 17

pSG-EP bla cat+

spoILAABC

4

pSGMU2 bla cat+ 8

pSGMU187 blacat+spoVA 5

pT1 blafl-on cat+spoIlAC Thisstudy

recognize thespoIIIGpromoter,^wefused lacZtospoIIIG in each of ^our strains, making them spoIIIG mutants, and measured

0-galactosidase

activityatintervals afterthecells wereresuspended in sporulation medium. Typical resultsare

shown inFig. 3. Strain M-7 initiated

0-galactosidase

synthe- sis alittle later (30to 60

min)

than thespo+ strain Kl but eventually accumulated as much enzyme. Strain M-8 syn-

thesized 1-galactosidase atonly 15to20% of thewild-type rate. Strains M-9 and M-11 gave no more than the back- ground activity of ,3-galactosidase.

Atabout the sametimeasspoIIIG is transcribed by EuF intheforespore, alkaline phosphatase is synthesized in the mother cell. Transcription of the gene for alkaline phos- phatase is believed to be due to a different form of RNA polymerase, EaE (6). However, some (particularly non-

sense) mutations in spoIL4Cprevent the synthesis of alka- line phosphatase, while other (particularly missense)muta-

tions in thesamegenepermit synthesis, albeittoareduced extent(34), and Stragier and Losick (31) have suggested that the formation of active o5E from its precursor (pro--E) dependsonthe activity of

e.

Wefoundthat in strainsM-7 and M-8 alkalinephosphatase

wasmadeatthesametimeafter resuspensionasin strain Kl and accumulated to 90 to 110% of the wild-type activity.

Strain

M-9 made 15 to

20%

and

M-11 made less than 10%

as much enzyme as the wild type.

Assuming that the

appear- ance

of alkaline phosphatase is

a measure

of transcription by

Eo-r,

these

results suggest

that

very

low

oF

activity is compatible

with

wild-type levels

ofu-

activity. (The

sum- mary

of results

in

Table

2

emphasizes the noncoordinate effect of the mutations

on

the three properties that

were

measured.)

One possible explanation

for

the results is that

^a

sigma factor that

lacks 9 or more

C-terminal residues is unusually sensitive

to

proteolytic degradation,

sothat its

concentration in sporulating cells is

too

low

to

sustain its normal function.

(Parsell

et

al. [22]

have shown

that in

A repressor the

unstructured region

near

the

C

terminus determines

suscep-

tibility

to

proteolytic cleavage.)

To test

this possibility,

we

used Western blotting

to measure

e1

inthe spo+ strain Kl

and in strains M-9

and M-11. We

found

no

difference

among the three

strains either

at

t1

or att4

(Fig. 4).

DISCUSSION

We

constructed

mutant

strains that synthesize C-trun- cated e proteins by introducing

nonsense

mutations into spoILAC.

Even

though the

nonsense

mutations lie

in

adja-

cent

codons, with the result that the corresponding proteins have 246, 247,

or

248 residues, the three

mutants

have

very

different phenotypes.

These

differences

cannotbe

explained by assuming

that

there is

anochre suppressor in

the genetic background which allows

some

synthesis of wild-type er

in

strains M-7 and

M-8:

the

two mutants are

phenotypically distinct (Table 2),

and

in

any case an

ochre mutation

elsewhere in the same gene

completely abolishes e activity

in an

isogenic strain (34).

We

therefore believe that

the results

reflect the activity

of the

eo1 protein after it has been truncated in these strains and thus contribute

to our

under- standing

of the function of the

C-terminal portion of

the

wild-type e.

Mutation

spoIIACQ249(Oc)

removes the

7 C-terminal amino acid residues.

A

strain containing this mutation

sporu-

lated normally and made wild-type quantities of alkaline phosphatase,

and

transcription

of

spoIHIG proceeded

at

the

same rate as in the wild type

(although

the initiation of

transcription

was

slightly delayed).

We conclude that the

final 7

of

the 8 residues of the C-terminal tail

can be

removed without much affecting the activity of e'.

Mutation spoIL4CK247(Am) deletes only

2

additional

res-

idues,

and yet the

resulting

mutant

sigma

factor was unable to

direct detectable transcription from spoHIIG.

The 2

amino acid residues missing from this strain but

present

in the

strain

just described

are

Lys-247 and Val-248. Lys-247 is the C-terminal residue of the basic region that lies adjacent

to

the helix-turn-helix motif of e, and

our results

therefore

indicate that the

activity

of theprotein is dependent on the

integrity

ofthis

region,

as one

might

expect

from its

conser- vation among

the

known

sigma factors.

It

is

tempting to

speculate

that this basic

region binds

viaelectrostatic inter- actions to DNA

(perhaps by folding

around the backof the double helix in a manner

akin

to

the

N-terminal arm of the

XCI

repressor

[20, 21])

andhelps to stabilize the interaction ofthe

helix-turn-helix

motifwith the -35 region of its target promoter.

The third of the mutations that we have studied,

spoII4CV248(0c),

removes

Val-248

but

leaves

intact Lys-

247,

which now

becomes

the C-terminal residue of the

protein.

The mutant

e

that results transcribed

spoIIIG

at

only

about

20%

of the

wild-type

rate. Wesuggest an expla- J. BAcTERIOL.

aF PROTEINS TRUNCATED NEAR THE C TERMINUS 7147

A 245

Lys Gln

250

Ile Lys Val Gln Met Asp His AAA CAG ATC AAG GTT CAA ATG GAT CAT

residue number

wild-type amino-acidsequence wild-type nucleotidesequence

AAA CAG ATC AAG GTT TAA

AAA CAG ATC AAG --- TAA

ATG GAT CAT ATG GAT CAT

M-7 nucleotidesequence

M-8 nucleotidesequence

AAA CAG ATC TAG GTT CAA ATG GAT CAT M-9 nucleotidesequence ScaI

i

l

spolIAC cat

4 linearizedby Scal

mutantplasmids

lII

spolIAC cat spoVA 5

chromosome ofrecipient strainK3 Transfomation andselection forCmR

-> spollAC cat spoVA

chromosome ofmutantstrains

FIG. 2. Construction ofmutantstrains. (A) Nucleotidesequenceof codons244to252 of wild-type andmutantspoILAC.The underlined residues are complementary to the mutant oligonucleotides used in the construction of strains M-7, M-8, and M-9 (see Materials and Methods). (B) Linearized plasmids whichcontain themutantspoIlACsequences arerecombined into the recipientstrain K3 bya double

crossoverbetween thehomologous regions,withselection for chloramphenicol resistance.Theresultingstrainshaveamutation inspolA4C and^arechloramphenicolresistant. An asterisk isusedtoindicate ^amutation.

'a)

-4

.104

0

C 10

._i

v)0^ow

O

^{0 Time1 after2initiation3 of}

sporulation (h)

^{4 5}

FIG. 3. Effect of mutations ^on the expression of the spoIIIG

geneinresuspendedcultures. StrainsKl(closed circle), M-7(open circle),M-8(closedsquare), M-9(closed triangle),and M-11 (open triangle)wereresuspendedattime 0.

P-Galactosidase

activitywas

assayedas a measureofspoIIIG-lacZ expression.

nation for

this

result in terms of our proposal that Lys-247 is involved in

electrostatic

interaction with the spoIIIG pro- moter;

it

may be that

placing

this residue at the -COO-

terminus

of

the protein hampers

its

interaction with

DNA. In

wild-type sigma

factors the basic

region is always separated

from the C-terminus

by

several

residues,

and these may serve as aspacer to remove the

positive charge of this region

from the

-COO- terminus. Despite the rather weak

tran-

scription of spoIIIG,

a

strain carrying

this mutant

o-F

can

sporulate normally.

It has been

suggested (32) that aJ activity

is necessary

only

to

begin transcription

of

spoIIIG relatively early

in

sporulation

and that

cr,

the

product

of

TABLE 2. Summaryofphenotypesof the mutantstrainsa

Mutantstrain Sporulation spoIIIGexpression Alkalinephosphatase

M-7 +++ +++b +++

M-8 +++ + +++

M-9 - - +

M-11 - - -

a+ ++, 90to110%ofwild-typesporulation incidence,spoIIIGexpression,

orenzymeactivity;+, 15 to20% ofwild-typesporulation incidence,spoIIIG expression,orenzymeactivity;-,nodetectablesporulation,spoIIIGexpres- sion,orenzymeactivity.

bExpressionwasdelayed by30to60 min.

VOL. 174,1992

7148 MIN AND YUDKIN

1 2 3 4 5 6

FIG. 4. Immunoblotting ofcell extracts with anti-of antibody.

Lanes: 1,strain Kl at

t,;

2,strain Kl at t4; 3, strain M-11 at

tl;

4, strain M-11 at t4; 5, strain M-9att1; 6,strain M-9att4. The arrow indicates the position ofpurified or marker.

spoIIIG, can then direct transcription of its ownstructural gene; indeed, o-F and aG are known to havevery similar recognition targets. If this suggestion is correct, it may be that transcription of spoIIIG bya mutant a-F atonly 15 to 20% of the wild-type rate is sufficient to startthe catalytic cascade and thusto achievea normal incidence ofsporula- tion.

ACKNOW1LEDGMENTS

K.-T.M. wasthe recipient of a scholarship fromI. C. I. Korea.

We aregratefultoJeffErrington for his valuablecomments onthe manuscript and to CorinneHilditch for her help with immunoblot- ting.

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