Biochimica et Biophysica Acta 842 (1985) 184-188
Elsevier BBA 22142
C o m p e t e n c e proteins in B a c i l l u s s u b t i l i s c o m m u t a n t s Claudia Barberio, Raffaella Coppolecchia, Giorgio Mastromei and Mario Polsinelli Dipartimento di Biologia Animale e Genetica, Universith di Firenze, v. Romana 17, 50125 Firenze (Italy)
(Received May 7th, 1985)
Key words: Competencemutant; DNA nuclease; Pleiotropiceffect;(B. subtilis)
The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these corn mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.
Introduction In bacterial cells, the transformation process goes through several steps common to most of the naturally transformable bacteria. One of the first passages is competence development: a physiological condition under which a cell can bind and take up transforming DNA. During competence development, many specific proteins are synthesized, several of which are involved in the processing of exogenous DNA [1,2]. Mutants impaired in competence development (corn mutants) can not be transformed, because they are unable to bind a n d / o r take up DNA. In Bacillus subtilis the synthesis of new proteins during competence development has been reported , but little is known about the genes involved and the biochemical nature of the products. An autolytic activity  and a competence factor  have been described and, recently, it has been reported that two different polypeptides, a DNAbinding protein and a nuclease, are necessary for DNA binding and entry . Although this information comes mainly from the study of mutants
altered in competence, the corresponding mutations have not been mapped so far; moreover, it is not known how many other proteins are involved in this process. In a previous paper we described the mapping of four different corn mutations . We now report data concerning the study of the corn mutants for the presence of competence-specific proteins.
Materials and Methods Strains. T h e B. subtilis strains PB19, PB3361 and the com mutants were previously described . Biochemicals. The electrophoresis reagents were purchased from Bio-Rad and BRL. 14C-Aminoacids were supplied by Amersham (spec. act., 56 mCi/matom). Preparation o f protoplast supernatants and m e m branes. Protoplast supernatants and membranes,
from 4-1 cultures, wer¢"prepared according to Mulder and Venema . Proteins were determined by the method of Lowry et al. . Labelling and extraction o f cellular proteins. Cells
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185 were grown to competence as previously described . 14C-Aminoacids were added at a final concentration of 1 #Ci/ml during the last 60 rain of cell growth to competence. Finally, sodium azide was added (50 #g/ml final concentration), and the culture was put on ice. The following steps were run at 4°C. 15 ml of the culture were centrifuged for 10 rain at 4000 × g. The pellet was washed twice with 20 ml of 20 mM Tris-HC1 (pH 7.5) containing 50 # g / m l sodium azide, and finally resuspended in 4 ml of 10 mM Tris-HC1 (pH 7.5) containing 5 mM MgCI 2. Cells were broken by sonication (three pulses of 1 rain each at full power) using a Branson sonifier (Model B-12). DNAase and RNAase were added at 50 #g/ml each, and the extracts incubated for 30 min at 4°C. The samples were frozen at -70°C, liophylized and resuspended in 0.4 ml of isoelectrophocusing buffer. 14C-Aminoacids incorporation was measured as trichloroacetic acid-precipitable counts. Nuclease activity detection. Nuclease activity was detected on SDS-DNA polyacrylamide gels as described by Mulder and Venema . Two-dimensional gel electrophoresis. Samples were run on two-dimensional gels according to the method of O'Farrell . In the first dimension, the ampholine pH range was 4-6.5; in the second, the acrylamide concentration was 15%. Isoelectrofocusing was performed loading 40 #g (25 #1) of membrane samples or 200000 cpm (50 #1) of total extracts. Proteins from membranes were visualized by silver staining, as described by Wray et al. , with the following modifications. Before staining, gels were soaked overnight in a solution of 30% methanol 12% trichloroacetic acid and then washed twice with distilled water (60 rain each time). The destaining step, in our case essential to eliminate a dark background, was performed using the fixing bath Agefix (Agfa) diluted 1:2, followed by a wash in 0.2% acetic acid. Proteins, present in total extracts, were stained with Coomassie brilliant blue. Gels were then treated with an autoradiography enhancer (EN3HANCE, New England Nuclear), dried and exposed at - 70°C to Kodak X-Omat films SO-282 for 20 days. Films were preflashed as described by Laskey and Mills .
Results and Discussion D N A nucleases in competence mutants The study of corn mutations has recently al-
lowed the identification of DNA nucleases and polypeptides specific for competence development in B. subtilis [12,13]. Based on these studies, a model has been proposed for DNA binding and entry in this bacterium . In our laboratory, we have isolated and characterized four different B. subtilis corn mutants. We showed that three of them (FB 91, FB 92, FB 93) were impaired in DNA binding, while the fourth (FB 94) was blocked in DNA entry . In order to correlate the four corn genes with their products, we examined the nuclease activities present in protoplast supernatants and in membrane preparations. This was done because Mulder and Venema  have shown that B. subtilis competent cells produce specific DNAases which are associated with the cytoplasmic membrane and partially released during protoplasting . The samples loaded on the gels were not heated in order to detect also nuclease activities due to protein complexes. The gel picture (Fig. la) shows that two mutants, FB 91 and FB 93, are strongly reduced in the 28, 17 and 14 kDa activities found in protoplast supernatants and they closely resemble the non-competent control. In the other two mutants, the enzyme activities are very similar to those of the wild-type competent cells. Since the reduction in nuclease activities could be due to lack of detachment from the membrane of these proteins, we measured the activities associated with the membrane. The membrane nucleases show patterns similar to those observed in protoplast supernatants (Fig. lb); in this case the 14 kDa band is completely absent in strains FB 91 and FB 93. The slight reduction of the same band in FB 92 could be non-significant, since variation in this membrane-bound activity has already been described . The above observations were confirmed when examining heated samples (data not shown), even though in this case we mainly observed bands at 14 and 17 kDa, since the heating process splitted all protein complexes. The 14, 17 and 28 kDa activities correspond to those described by Smith et al.  as type I, II and III of which the last one is involved in DNA entry
 and is part of a 75 kDa complex responsible for D N A binding. The very low activities found in the FB 91 and FB 93 strains might indicate the presence of an incomplete 75 kDa complex and explain why these mutants are binding deficient . On the other hand, this complex could be present, but blocked in some of its functions, in the FB 92 and FB 94 strains, which have a wild-type level of nuclease activities. This may also explain why the potassium phosphate buffer partially restores the transformation capacity in these last two mutants , if we postulate that the buffer removes the block which impedes D N A uptake by the 75 kDa complex. Competence proteins in membranes Many proteins synthesized during competence development and responsible for D N A binding and entry are associated with the membrane. For this reason we studied the effect of the four corn mutations on the synthesis of these membrane proteins. Membranes were isolated from competent and non-competent cells, and the proteins were separated by two-dimensional gel electrophoresis. Competence-specific proteins were identified by comparing the patterns obtained with membranes from competent (Fig. 2a) and non-competent (Fig. 2b) cells of the parental strain PB 3361. In Fig. 2a, the open circles mark the 12 competence polypeptides identified by this kind of analysis, while the arrows point to the spots missing in at least one of the mutants.
Fig. 1. Nuclease patterns of wild type and mutant strains in DNA containing SDS-polyacrylamide gels. Samples are from (a) protoplast supernatants and (b) membranes of competent cells; * wild-type cells collected half-wayduring the exponentialgrowth phase (non-competent).Samples(150/~g of proteins each) were not heated before loading on the gel. Gels were incubated for 72 h at 37°C in the presenceof MnC12. The polypeptide number 1 (pI 5.6; 32 kDa) is not present in FB 92, while the number 2 (pI 5.7; 43 kDa) is absent in FB 91 and FB 93. The mutant FB 94 has all the 12 polypeptides. In the strains where a polypeptide is missing, we have been looking for new spots, which could be generated by the mutated protein, but we did not detect such spots. The lacking polypeptides are probably not correlated with the previously discussed D N A nucleases, because they have a different molecular weight. Furthermore they might not even be the products of the mutated corn genes. In fact, the spot number 2 is absent both in FB 91 and FB 93, which are known to carry two different mutations . FB 92 is missing the polypeptide number 1, which might be the product of the com-71 gene, but this is not supported by any other experimental data and, as discussed in the next section, many more spots are absent in this strain, when competence proteins were studied in total cellular extracts. Competence proteins in total cellular extracts To study the effect of corn mutations on total protein synthesis, we separated 14C-labelled competence proteins by two-dimensional gel electrophoresis. In the analysis of the autoradiographies we concentrated our attention on 80 polypeptides which appeared to be welI labelled in the wild-type cells. For this reason we did not consider some polypeptides identified as specific of competence in membrane preparations. Autoradiographs of the
Fig. 2. Two-dimensional eleetrophoretic analysis of membrane associated proteins from competent (a) and non competent (b) cells of the wild type strain PB 3361. The open circles (a) mark the polypeptides found only in the competent sample. The spot number 1 is missing in strain FB 92, the number 2 in strains FB 91 and FB 93.
gels of the parental strain PB 3361 and of the mutant FB 91 are shown in Fig. 3. The arrows mark the position of the eight spots present in PB 3361 but absent in FB 91. A similar comparison has been done with the other three strains; the data, concerning the polypeptides missing in at least one mutant, are summarized in Table I. These data show that two polypeptides are missing in all the mutants, and another six in three of the four strains. Again FB 93 shows the same pattern of FB 91, except that it is missing two more proteins, and it could be that one of the two is the product of the corn 93 gene.
Fig. 3. Two-dimensional electrophoretic analysis of 14C-labelled competence proteins from (a) the wild type strain PB 3361 and (b) the mutant strain FB 91. The arrows (a) mark the polypeptides that are missing in the mutant preparation.
The pI 5.17, 19 kDa polypeptide is certainly the binding unit of the 75kDa complex described by Smith et al.  and already discussed. This polypeptide is missing in all the mutants, except in FB 92 (Table I) where, anyhow, it is inactive since the strain is binding deficient . On the contrary, the binding activity of the FB 94 strain  could be due either to a different protein or to a modified form of the same polypeptide, even though we have not been able to detect such a new spot on the gel. The pI 5.30, 47 kDa polypeptide has a position indistinguishable from the recE gene product, which is predominantly present in competent cells , Its absence in two of the mutants (Table I)
188 TABLE I
way. We tend towards the last hypothesis, but with
the approach used we have not been able to work out the order in which competence proteins are
+ indicates the presence and - the absence of a specific polypeptide. The other 63 competence proteins are present in all the mutants.
synthesized. We are presently trying to overcome this problem by cloning the com genes and trying to understand in this way which role they play in competence development.
4.23 4.93 4.93 5.17 5.17 5.30 5.30 5.30 5.43 5.51 5.55 5.55 5.75 5.82 5.87 5.87 6.19
26 41 49 19 45 28 42 47 27 45 30 33 45 43 35 36 50
+ + + + . + . + + + +
+ + + + + +
+ + + + -
+ + + +
. + -
may be due to the low competence level reached b y t h e s e s t r a i n s , a n d / o r , as d i s c u s s e d b e l o w , t o t h e l a c k i n g o f a p r o t e i n n e c e s s a r y f o r its s y n t h e s i s . From the data on nuclease activities and two dimensional-gel electrophoresis it becomes clear t h a t all t h e c o m m u t a t i o n s w e h a v e s t u d i e d s h o w a s t r o n g p l e i o t r o p i c effect. T h i s m i g h t o c c u r e i t h e r i f t h e m u t a t e d g e n e s h a v e a r e g u l a t o r y f u n c t i o n o r if they code for initial products in a metabolic path-
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