J. nob. Uiol. (1’375) 96, 345-35” I

Messenger Ribonucleic Acid Content of Bacillus arnyloliquefaciens throughout its Growth Cycle compared with Bacillus subtilis 168 KBROWN Department

of &ochemistry, Clifton Boulevard,

AND G.C~LEMAN

University Hospital Nottingham

and Medical NG7 ZUH, England

8chool

(Received 7 November 1974, and in reuised form 12 May 1975) The messenger RNA contents of Bacillus amyloliquefackns and B. subti1i.s 168, grown in a 1% maltose-O~5~0 casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both csses there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subt&s this level was maintained into the stationary phase. By contrast, in B. amylo1iputfacien.s the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by dete rmining the relative rates of incorporation of L-[14C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each speoies, remaining at a constant low level in B. subtilk and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefacie~, no evidence was obtained for the aooumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.

1. Introduction A model has been proposed for the regulation of extracellular enzyme Bacillus amyloliquejaciens, a process which occurs almost exclusively

formation

in

in the postexponential phase of the growth cycle, based on competition at the level of RNA synthesis (Coleman et al., 1974). According to this model two separate effects, one of which may be superimposed on the other, are considered to be responsible for a post-exponential increase in exoprotein mRNA. The fist is an increase in availnblc RNA polymerase on “switching off” ribosomal RNA synthesis and the second an increase in substrate concentration resulting from rRNA turnover. This is not consistent with the findings of Both et al. (1972) and Gould et al. (1973), who obtained indirect evidence based on the results of inhibitor studies that exoenzyme-specific mRNA accumulated within the cells of B. amyloliquejacie~~ in vast excess, capable of supporting exoenzyme formation for a prolonged period. These authors further suggest that a protease mRNA pool was present when cell growth was proceeding rapidly. 345

S. HltO\VIU A\NL, G. COLEMAN

:; Iii

The prcswt

an attvrnpt t,o distinguish bct~wucv~ the t\vc~ mutlt& I,>, RNA Icvels throughout t’hc growth qxlo of U. avra!lloEiquefaciev~s and to make a direct comparison with the closely-related organism B. subtilis 168, which ha’s been the subject of somewhat related studies (Midgley, 1969; Semet,s et al., 1973).

direct

study represents

measurements

of

messenger

2. Materials and Methods (a) Ovyaniavn8

U. w,,lyloliqcLefacie7kY strain

T (Welker

I!& Campbell,

(b) G’rowth uf bacterial J3ut,h urganisms medium and under

\va.s osLimatetl

1Sacl~urial dt:llsiL,ios

‘I’llu total RNA (Schnaidor, 1957).

as described

\vuru duterminocl

culltont

S: Elliott,

by 11x method

(f) l~-[U-14C]aaline

culls

1iydrulysuLo

complex

estimation

by Coleman

of bacturial

lti8 were usucl.

cultm-es

were grown in the I’>; n~altoso---0.5°/o cilsoin the conditions described by Coleman (1967).

(0) cc-Amyhe u-Amylaso

lY67) and U. 8ublilis

was

(lY62).

of Sturmunt,h

tlcl.c:rminocl

by

d Coleman

the

urcinul

(1!17Y).

method

iwm-puration

111 urder to determine tile rate of incorporation of [14C]valinc into bacterial proteins 10 &i of L-[U-14C]valine was added to 10 ml of culture contained in a 100 ml conical flask and the preparation was incubated with shaking at 30°C. Samples (1 ml) were taken at the time of addition of the radioactive amino acid and at 5-min intervals thereafter, oath being placed in a centrifuge tube and rapidly cooled to 0°C. Each cooled preparation \vas centrifuged for 3 min at 5000 g. The supernatant fract,ion was retained and the pellet washed twice with ice-cold 0.9% lic’l. The washed pellet wae then dispersed irk whilst a 0.5 ml ;! ml of 5”/: (w/v) trichloroacet,ic acid containing 0.1 y& caseill hydrolysate sample of the supernatant, fraction W-W added to 0.5 ml of lOo/o (w/v) trichloroacetic acid ‘I’lle preparat,ions were then heated in a water batlr containing O.Zq& casein hydrolysate. ilt 9O’C for 30 min. Subsequent treatment ww as described by Coleman SCElliott (I 965).

(g) lCantlo,nly labelled RNA preparath ‘I’110 bacterial presence of 1 l&i t,hat at the time for a furt.her 1 -h as described by culunm step was

cultures were labelled by growing over at least fuur cell divisions in tllcb [2-14C]ura.cil/ml (spec. radioact. 1 &i/60 pg). Care was taken to ensure of harvesting sufficient uracil was still present to allow incorporation period. The randomly labelled RNA was isolated from the bacterial cells Pigott & Midgley (1968) with hhe exception that the Sephadex G200 omitted.

‘l’he hybridization procedure was that dcacribed by Pigutt & Midgley DNA:RNA ratio of 10: 1 wa8 used unless ot,herwise stated. It is important only mRNA will bind at the DNA:RNA ratios used during t’he llybridization procedure represents a valid measure of mRNA.

(l!K%) al~d a to llot,e that and so the

(i) Radiochemicals The [2-14C]uracil (spec. radioact. 280 mCi/mmol) used were obtained U.K.

61 mCi/mmol) and L-[U-14C]valine (spec. radioact. from The Radiochemical Centre, Amersham, Bucks.,

3. Results (a) Relation between total cellular messenger RNA and extracellular a-amylase during bacterial growth B. amybldquefaciens and B. subtilis 168 were grown under identical

conditions at 30°C in a I “/o maltoseXG% casein hydrolysate medium in the presence of 14C-lal~elled uracil. Samples of the cultures wcrc taken at different times throughout the gron-tlr cycle for bacterial density determination. The supernatant fraction of each sample was assayed for a-amylase. Amylase was chosen as a convenient marker for extracellular protein since, like protease, its formation exactly parallels total exoprot(ein under the conditions used (Coleman, 1967 ; Stormonth & Coleman, 1974). The bacterial pellets corresponding to the supernatant fractions were retained and randomly labelled RNA was isolated from each. The percentage of the radioactivity in these RNA preparations which hybridized at a DNL4 : DNB rat,io of IO : 1 was determined as a measure of total messenger RNA.

lncubotlon

time

(h) lb)

Fra. 1. Relation ( --e--a--)

during

between total cellular messenger RNA (-O-O-) the growt,h ( ---A----A-) of (a) H. nrnzlloZigvrf/rci~)l~

and extracellular a-amykr and (b) 13.subtiZi8 168.

The results are shown in Figure 1 where it, ran be seen that, both organisms had very closely similar growth characteristics. However, whilst B. nmyZoZiqut$wienn produced little a-nmylase during the exponential-phase, enzyme formation increased to a high linear rate after exponential growth ceased. In this same organism the messenger RNA accounted for a constant proportion of the total RNA during exponential growth. After the end of exponential growth, mRNA increased reaching a level tn-ic>e that present in exponential-pha,se cells when exoenzymo secretion was maximal (Fig. 1(a)). By rontrast, the messenger RNA of B. suhtilia 168 represented a constant) proportion of t)hr total RNA t#hroughout the growth cycle, equal t,o that in exponential phase B. wmyloliqwfaciens. a-Amylase was secreted at a very low and apparentl) constant, rat)e nvcr the whole perind st,udirtl (Fig. I (I))). (1))

Relative rake of Cncorporatiow 01 I,-[ U-“C’]valine into cellular and extracellular protein

In order to resolve the total messenger RNA into two components, that is, that roncerned Aith cell protein produrtion and t’hat neressa’ry for the formation of

S. BROWN

348

AND

G. COLEMAN

exoprotein, an assumption was made that the messenger RNA for each fraction was translated with equal eficiency at each point examined during the growth cycle. In this event then the relative amounts of the two mRNA species will be equal to the relative rates of synthesis of the two protein fractions. As a measure of the latter L-[UJ4C]valine was added to samples of culture and the relative rates of incorporation of the radioactive amino acid into cellular and extracellular protein, over a short t’ime period, was determined, in each case. (There is no significant difference between the valine contents of cell and extracellular protein.) The results are shown in Table 1, where it can be seen that in B. amyloliquefaciens the rate of labelled amino acid incorporation into cell protein during the exponential phase was 970/, of the total. After the end of exponential growth, extracellular protein accounted for an increasing proportion of the radioactivity incorporated. When the stationary phase was reached at 28 hours, very nearly equal rates of incorporation were observed into the two protein fractions. In the case of B. szGbtilis 168 the relative rates of incorporation werr constant throughout the growth cycle, the rate of incorporation int’o cellular protein accounting for 95% of the total. (c) Total RNA contents of B. amyloliquefaciens and B. subtilis 168 throughout their growth cycles In order to be in a position to make a more meaningful comparison between the results of the hybridization studies expressed in Figure 1, which show messenger RNA as a percentage of total RNA, measurements were made of the RNA contents of the two organisms throughout their growth cycles. It can be seen in Figure 2 that the RNA contents of both species were constant and closely similar throughout the exponential phase of growth. During the subsequent lag phase there was a reduction in total cellular RNA. On reaching the stationary phase the levels were 19% and 17 y. less than the exponential phase values in B. amyloliquejaciens and B. subtilis 168, respectively.

a z f

70

c'6

6C

I 18

I

I 22 lncubotion

Fro. 2. Changes in total RNA and B. sublilie 168 (-m-O-).

content

I

I 26

I

1 3( I

time( h)

during the growth

of B. amylaliquefnciene

(-O-O-)

BACTERIAL

MESSENGER

349

RNA

(d) Changes in cell protein messenger RNA and exoprotein messenger RNA throughout the growth cycle Using the data expressed in Figure 2 the percentage values for the messenger RNA content of the bacterial cells shown in Figure 1 were translated into actual amounts, expressed relative to unit mass of cellular material. Further, using the results in Table 1 the values for the total messenger RNA were, in turn, resolved into cell protein mRNA and exoprotein mRNA components. The results in Figure 3(a) show that in B. anayloliquefaciens there was a low level of exoprotein mRNA in the exponential phase of growth consistent with the low level of a-amylase secretion shown in Figure l(a). However, at the beginning of the post-exponential phase, when the amount of exoprotein mRNA started to increase, there was a parallel increase in the rate of exoenzyme secretion. Exoprotein mRNA levelled off at its highest value at the beginning of the stationary phase when a high linear rate of u-amylase production was achieved. By contrast, again consistently with the characteristics of u-amylase secretion, B. subtilis 168 was shown to have a lnw and ronsiant level of exoprotein mRNA throughout the growth cycle (Fig. 3(b)). TABLE

1

Relative rates of incorporation of L-[U-‘b]vdine into cellular and extracellular protein at diflerent stagesduring the baLteria1 growth cycle Relative Organism

Incubation time (4

rates of L-[U-14C]valine incorporation (O/o of total) Cellular Extracellular prot,ein protein

B. amylol~wefaciens

17 20 23 26 28 30

97.5 97 77.6 66 62-B 52-5

B. eubtilia 168

18 26 30

96 93 96

2.8 3 22.8 34 47.8 47.6 5 7 5

In keeping with the similar characteristics of growth, the cellular protein mRNA contents of both B. am.yJoliquefaciens and B. subtilis 168 were closely similar and remained at 2 to 3 pg/mg cell material throughout their growth cycles. (e) aerial hybridizution

of B. amyloliquefaciens

messenger RNA

If an accumulation of exoprotein mRNA occurred to such an extent that it was capable of more than saturating all the sites on the DNA during hybridization an erroneously low level would be detected. With this in mind a serial hybridization experiment was carried out in which the randomly labelled RNA, isolated from both exponential-phase and post-exponential phase bacteria, which did not become

‘Iai ! .A---*---*-*-. I I

22

26 (a)

30 ia Incubation

I

time(h)

26

30

(b)

FIO. 3. Relrkive tlintribution of cellular protein mRNA (--o-O---) mRNA (--a-@---) during the growth cycles of (a) B. amyloliq~tefacirns

Number of hybrldizallon

-,

I

22

and extracellular protein and (b) R. twhfilie 168.

events

FIQ. 4. Serial hybridization of B. nmy2oZiquefnciena messenger RNA. Preparations of randomly labelled RNA isolated from exponential-phase (0) and post-exponential phase ( l ) bact,eria were serially hybridized with DNA as described in the t)axt.

attached to DNA, under the conditions of the hybridization procedure, was hybridized with more DNA. The process was repeated until no more RNA hybridizable at a DNA : RNA ratio of 5 : 1 remained. The rationale was that any mRNA species which was limiting at the first incubation with DNA would be hybridized to a diminishing extent, but with the same efficiency, during succeeding steps. Concomitantly the same amount of any mRNA species present in excess would become attached to complementary DNA sequences at, every stage at which it remained in excess. Thus, when

BAc’rEltlAL

I\IESSII:&C;Eli

11x.\

3.7I

comparing different RNA preparations an excess of a particula,r mRNA species in one preparation would lead to increasingly divergent values as the rchybridizat,ion process was continued. This latter clearly does not apply: since, as shown in Figure 4, the serial hybridization curves for the two RNA preparations were almost identical. If an accumulation of particular mRNA components had occurred at both stages of grouth examined then the serial hybridization curves might also be identical. However, the fact that the hybridization occurs with the same efficiency at each step supports bhc idea that there is no accumulation of a, particular spccica in large quantity.

4. Discussion ln B. subtilis 168 the mRNA content accounts for a constant proportion of the total cellular RNA throughout the growth cycle. By contrast, in B. amyloliquejaciens which exhibits similar growth charact,eristics, the mRNA undergoes a twofold increase aft#er t,he end of exponential growth. This post-exponential increase in B. amybliquefacierrs mRNA is considered to be implicated in the massive exoprotcin formation which occurs in the same phase. That this is a rcasonablc conclusion is supported by thts observatiou that when the total messenger RNA of both organisms is resolved int,o two components, the cxoprotein mRNA portion in each case bears the expected relationship to the observed patterns of exoenzyme secretion. Further, no significant difference was found between the cell protein mRNA levels in the two species, which is consistent with the similarities in their growth patterns. It should be noted that this relation can only exist if the cell protein mRN14 and exoprotein mRNA fractions are each translated with equal efficiency at every individual point in time throughout the growth cycle, even though the overall efficiency of translation becomes less as t)he culture passes into the post-exponential phase. This observation, therefore, provides compelling evidence in support of the validity of the only assumption which was made in this work. However, irrespective of whether the exoprot’ein mRNA is considered to be translated with greater, the same, or less efficiency than cell prvtein mRNA, the overall characteristics of change of each mRNA component,, in both organisms, is the same throughout the growth cycle and only their amount,s relat,ivc to each other change. The similarity in the serial hybridization patterns between exponential-phase mRNA, containing 25 times more cell protein mRNA than exoprotein mRNA, and post-exponential phase mRNA, containing equal amounts of the two component,s, would lend additional support to the present findings. This is contrary to the mechanism involving a vast excess of mRNA queueing up for translat,ion-extrusion sites as proposed by Both et al. (1972) and Gould et al. (1973). Further, the fact that there were DNA &es available for an amount of exoprotein mRNA at least equal to that detected in t,he stationary phase which, nevertheless. were not occupied during the hybridization of exponential-phase mRNA does not support the suggestion of Both et al. (1972) that’ a pool of cxoprotein mRNA m>r? accumulate during rapid growth. The authors wish to express their gratitude to Dr J. E. M. Midgley, who gave his time to introduce them to the more esoteric aspects of the DNA-RNA hybridization technique. One of us (S. B.) wishes to thank the Science Research Council for the award of a Research Studentship.

35”4

K. lS-;HOW’N

AND

U. COLEMAN

REFERENCES Both, G. W., Mclnnes, J. L., HanIon, J. E., May, B. K. & Elliott, W. H. (1972). J. Mol. BioE. 67, 199-217. Coleman, G. (1967). J. f&z. Mkrobiol. 49, 421-431. Coleman, G. & Elliott, W. H. (1962). B&hem. J. 83, 256-2ti3. Coleman, 0. & Elliott, W. H. (1965). Biochem. J. 95, 699-706. Coleman, G., Brown, S. & Stormonth, D. A. (1974). J. Gen. Microbid. 81, viii-ix. Gould, A. R., May, B. K. & Elliott, W. H. (1973). J. Mol. Biol. 73, 213-219. Midgley, J. E. M. (1969). Biochewa. J. 115, 171-181. Pigott, C. H. & Midgley, J. E. M. (1968). Hiochem. J. 110, 251-263. Schneider, W. C. (1957). Meth. Enzymol. 3, 680-684. Semets, E. V., Glenn, A. K., May, B. K. & Elliott, W. H. (1973). .J. f~acteriol. 116, 531L534. Stormonth, D. A. & Coleman, C. (1972). J. Cen. Microbial. 71, 407.-408. Stormonth, D. A. 8s Coleman, G. (1974). J. appl. Bacterial. 37, 225-237. Welker, N. E. & Campbell, L. L. (1967). J. Bacterial. 94, 1124-1130.