Biomedical subjects

D Dubnau

Publications and source records attributed to D Dubnau.

At least 55 records · Page 3Linked to original sources

PubMed1991

Genetic competence in Bacillus subtilis.

Genetic competence may be defined as a physiological state enabling a bacterial culture to bind and take up high-molecular-weight exogenous DNA (transformation). In Bacillus subtilis, competence develops postexponentially and only in certain media. In addition, only a minority of the cells in a competent culture become competent, and these are physiologically distinct. Thus, competence is subject to three regulatory modalities: growth stage specific, nutritionally responsive, and cell type specific. This review summarizes the present state of knowledge concerning competence in B. subtilis. The study of genes required for transformability has permitted their classification into two broad categories. Late competence genes are expressed under competence control and specify products required for the binding, uptake, and processing of transforming DNA. Regulatory genes specify products that are needed for the expression of the late genes. Several of the late competence gene products have been shown to be membrane localized, and others are predicted to be membrane associated on the basis of amino acid sequence data. Several of these predicted protein sequences show a striking resemblance to gene products that are involved in the export and/or assembly of extracellular proteins and structures in gram-negative organisms. This observation is consistent with the idea that the late products are directly involved in transport of DNA and is equally consistent with the notion that they play a morphogenetic role in the assembly of a transport apparatus. The competence regulatory apparatus constitutes an elaborate signal transduction system that senses and interprets environmental information and passes this information to the competence-specific transcriptional machinery. Many of the regulatory gene products have been identified and partially characterized, and their interactions have been studied genetically and in some cases biochemically as well. These include several histidine kinase and response regulator members of the bacterial two-component signal transduction machinery, as well as a number of known transcriptionally active proteins. Results of genetic studies are consistent with the notion that the regulatory proteins interact in a hierarchical way to make up a regulatory pathway, and it is possible to propose a provisional scheme for the organization of this pathway. It is remarkable that almost all of the regulatory gene products appear to play roles in the control of various forms of postexponential expression in addition to competence, e.g., sporulation, degradative-enzyme production, motility, and antibiotic production. This has led to the notion of a signal transduction network which transduces environmental information to determine the levels and timing of expression of the ultimate products characteristic of each of these systems.

Amino Acid Sequence↗

PubMed1990

Binding of Bacillus subtilis ermC' methyltransferase to 23S rRNA.

ermC 23S rRNA methyltransferase dimethylates adenine 2085 in Bacillus subtilis 23S rRNA and also regulates its own synthesis by autogenous translational repression. We have characterized the binding of ermC' methyltransferase to 23S rRNA. This protein differs in only five amino acid residues from the ermC product and was chosen for study because of its greater stability and ease of isolation. A filter binding assay was used to study the physical aspects of binding in the absence of methylation. The dissociation equilibrium constant of the binding was found to be 4 x 10(-9) M at 37 degrees C. Kinetic studies of complex formation and dissociation revealed that the kon and koff were 4 x 10(6) M-1 s-1 and 6.8 x 10(-2) s-1 respectively at 16 degrees C. Equilibrium competition experiments showed that the enzyme has varying affinities for a variety of nucleic acids in the order 23S rRNA greater than 16S rRNA greater than M13 DNA, f2 RNA greater than tRNA. One of the end products of methylation, methylated 23S rRNA, had an affinity for the ermC' methyltransferase similar to that of unmethylated 23S rRNA. The binding affinity to 23S rRNA and the kinetics of the interaction were not detectably affected by the presence of AdoMet. The binding of ermC' methyltransferase to 23S rRNA had an unfavorable van't Hoff enthalpy (delta H = +6.2 kcal mol-1) and was driven by entropy (delta S = +56.2 cal mol-1 deg-1). The interaction between the two ligands involved at most two to three ionic pairings, and nonelectrostatic interactions contributed approximately 85% of the binding energy. The structural aspect of the interaction was investigated by probing with dimethyl sulfate, for ermC' methyltransferase dependent protection of 23S rRNA. A region of protection was detected, in the vicinity of the central loop of rRNA domain V and surrounding the site of methylation.

Autoradiography↗

PubMed1990

A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems.

A Bacillus subtilis gene, required for genetic competence, was identified immediately upstream from the previously characterized gene comA. The comA gene product has been found to exhibit amino acid sequence similarity to the so-called effector class of signal-transduction proteins. DNA sequencing of the new determinant, named comP, revealed that the carboxy-terminal domain of the predicted ComP protein is similar in amino acid sequence to that of several sensor members of the bacterial two-component signal-transduction systems. The predicted amino-terminal domain contains several hydrophobic segments, postulated to be membrane-spanning. In vitro-derived comP disruptions are epistatic on the expression of all late competence genes tested, including comG, comC, comD, and comE, but not on expression of the early gene comB. Although comA has its own promoter, some transcription of comA, especially later in growth, occurs via readthrough from comP sequences. A roughly twofold epistatic effect of a comP disruption was noted on the downstream comA determinant, possibly due to interruption of readthrough transcription from comP to comA. Overexpression of comA fully restored competence to a comP mutant, providing evidence that ComA acts after ComP, and consistent with a role for the latter protein in activation of the former, possibly by phosphorylation. ComP probably is involved in transmitting information concerning the nutritional status of the medium, particularly the presence of nitrogen- and carbon-containing nutrients. ComP was also shown to play a role in sporulation, at least partly interchangeable with that of SpoIIJ, another putative sensor protein.

Amino Acid Sequence↗

PubMed1990

A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis.

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.

Alleles↗

PubMed1990

Identification of cis-acting sequences required for translational autoregulation of the ermC methylase.

ermC methylase gene expression has been shown to be limited by translational autorepression, presumably due to methylase binding to ermC mRNA. It was found that this repression occurs in trans, yielding a 50% reduction in translation of an ermC-lacZ fusion mRNA. We investigated the ermC mRNA sequences required for translational repression in vivo. A series of deletions identified sequences in the 5' regulatory region that were required for translational repression. These included sequences of the 5' stem-loop structure that were not required for induction, as well as some that were required. The implications of these results for regulation are discussed.

Bacillus subtilis↗

PubMed1990

Growth medium-independent genetic competence mutants of Bacillus subtilis.

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.

Bacillus subtilis↗

PubMed1990

Suppression of early competence mutations in Bacillus subtilis by mec mutations.

Although competence normally develops only in glucose-minimal salts media, mecA and mecB mutations permit the expression of competence and of late competence genes in complex media as well (D. Dubnau and M. Roggiani, J. Bacteriol. 172:4048-4055, 1990). The expression of late competence genes is dependent on the products of the regulatory genes comA, comB, comP, sin, abrB, spo0H, and spo0A. We show here that this list must be extended to include degU, csh-293, and spo0K. mecA and -B mutations bypass most of these requirements, making the expression of late competence genes and of competence itself independent of all of these regulatory genes, with the exceptions of spo0A and spo0K (in the case of mecB). The expression of late competence genes in mec mutants that are deficient for each of the bypassed regulatory functions is still under growth stage-specific regulation. The implications of these findings are discussed, and a provisional scheme for the flow of information during the development of competence is proposed.

Bacillus subtilis↗

PubMed1990

Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis.

comC specifies a protein product that is required for genetic competence in Bacillus subtilis. The probable transcriptional start site of comC has been localized by high-resolution primer extension analysis and shown to be preceded by an appropriately positioned sequence that resembles the consensus promoter for the sigma A form of RNA polymerase. Low-resolution S1 nuclease transcription mapping was used to identify the comC terminator, which is located near a palindromic element recognizable in the DNA sequence. Deletion analysis of the sequence upstream from the likely promoter identified a region required in cis for the expression of comC. An overlapping, and possibly identical, sequence was shown to inhibit the expression of competence and of several late competence genes, when present in multiple copies. This was interpreted as due to the titration of a positively acting competence transcription factor (CTF) by multiple copies of the promoter-bearing fragment. In crude lysates of B. subtilis grown to competence, a DNA-binding activity that appeared to be specific for the comC promoter fragment was detected by gel retardation assays. This activity, postulated to be due to CTF, was detected only following growth in competence medium, only in the stationary phase of growth, and was dependent on the expression of ComA, a known competence-regulatory factor. In the presence of the mecA42 mutation, the ComA requirement for CTF activity was bypassed, and CTF activity could be detected in lysates prepared from a strain grown in complex medium. This behavior suggested that either the expression or the activation of CTF was regulated in a competence-specific manner. Comparison of the putative CTF-binding site defined by deletion analysis with a similarly positioned sequence upstream from the start site of the late competence gene comG revealed that both sequences contained palindromes, with 5 of 6 identical base pairs in each arm. It is suggested that these palindromic sequences comprise recognition elements for CTF binding and that CTF binding must occur for the appropriate expression of late competence genes.

Bacillus subtilis↗

PubMed1989

Mono- and dimethylating activities and kinetic studies of the ermC 23 S rRNA methyltransferase.

The ermC 23 S rRNA methyltransferase converts a single adenine residue to N6,N6-dimethyladenine, both in vivo and in vitro. The ermC methyltransferase was demonstrated to produce both N6-mono and N6,N6-dimethylated adenine residues in Bacillus subtilis 23 S rRNA during the course of the reaction in vitro. An almost total conversion of monomethylated intermediates into dimethylated products was observed upon completion of the reaction. Data presented here demonstrate that the addition of the two methyl groups to each 23 S rRNA molecule takes place through a monomethylated intermediate and suggest that the enzyme dissociates from its RNA substrate between the two consecutive methylation reactions. The enzyme is able to utilize monomethylated RNA as substrate for the addition of a second methyl group with an efficiency approximately comparable to that obtained when unmethylated RNA was the initial substrate. Initial-rate data and inhibition studies suggest that the ermC methylase reaction involves a sequential mechanism occurring by two consecutive Random Bi Bi reactions.

Bacillus subtilis↗

PubMed1989

Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis.

comC is a Bacillus subtilis gene required for the development of genetic competence. We have cloned a fragment from the B. subtilis chromosome that carries comC and contains all the information required to complement a Tn917lac insertion in comC. Genetic tests further localized comC to a 2.0-kilobase HindIII fragment. Northern (RNA) blotting experiments revealed that an 800-base-pair comC-specific transcript appeared at the time of transition from exponential to stationary phase during growth through the competence regimen. The DNA sequence of the comC region revealed two open reading frames (ORFs), transcribed in the same direction. The upstream ORF encoded a protein with apparent sequence similarity to the folC gene of Escherichia coli. Insertion of a chloramphenicol resistance determinant into this ORF and integration of the disrupted construct into the bacterial chromosome by replacement did not result in competence deficiency. The downstream ORF, which contained the Tn917lac insertion that resulted in a lack of competence, is therefore the comC gene. The predicted protein product of comC consisted of 248 amino acid residues and was quite hydrophobic. The comC gene product was not required for the expression of any other com genes tested, and this fact, together with the marked hydrophobicity of ComC, suggests that it may be a component of the DNA-processing apparatus of competent cells.

Amino Acid Sequence↗

PubMed1987

An in vitro study of the translational attenuation model of ermC regulation.

We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.

Bacillus subtilis↗

PubMed1987

Demonstration of erythromycin-dependent stalling of ribosomes on the ermC leader transcript.

ermC encodes a methylase that modifies 23 S rRNA, conferring resistance to macrolide-lincosamide-streptogramin B antibiotics. The expression of this gene is induced by erythromycin using a translational mechanism. We have employed the inherent RNase activity of a Bacillus subtilis S-30 extract as a probe for studying the interaction of ribosomes with ermC mRNA in the presence of antibiotics. 5' end-labeled ermC runoff transcript is a substrate for this RNase activity, while the ribosome-bound region of the RNA appears to be protected. Erythromycin- and oleandomycin-dependent protection of fragments of length 79-81 was observed during the translation of end-labeled ermC transcript. This occurs only using unmethylated (erythromycin sensitive) ribosomes. Various other antibiotics including clindomycin, tylosin, and lincomycin do not show this specific protection. These effects parallel the in vivo specificity of ermC induction. The effect of erythromycin can be abolished by using oligonucleotides complementary to regions of the ermC transcript upstream from nucleotide 71 and not by using an oligonucleotide complementary to a region of ermC downstream from that position. These results are interpretable in terms of the translational attenuation model and demonstrate that erythromycin-bound ribosomes initiate translation of the leader peptide, stall upstream from nucleotide 80 on the ermC mRNA, and thus make the ribosome-binding site for methylase message available for ribosome interaction.

Anti-Bacterial Agents↗

PubMed1987

Induced mRNA stability in Bacillus subtilis.

We have investigated the induced stability of mRNA encoded by the ermC gene in Bacillus subtilis. Induction of ermC gene expression by erythromycin is known to occur at the translational level. We show that this induction is accompanied by an increase in ermC mRNA half-life from about 2 min to about 40 min. Induced stabilization of ermC mRNA occurs independently of induced translation. The regulatory sequences required for stability are promoter-proximal and can confer induced stability on large mRNAs having diverse 3' ends. Translation of the ermC leader peptide and ribosome-stalling in the leader peptide sequence are necessary for induced stabilization.

Bacillus subtilis↗

PubMed1987

Cloning and analysis of ermG, a new macrolide-lincosamide-streptogramin B resistance element from Bacillus sphaericus.

To analyze the regulation of a newly discovered macrolide-lincosamide-streptogramin B resistance element (ermG) found in a soil isolate of Bacillus sphaericus, we cloned this determinant and obtained its DNA sequence. Minicell analysis revealed that ermG specifies a 29,000-dalton protein, the synthesis of which is induced by erythromycin. S1 nuclease mapping was used to identify the transcriptional start site. These experiments demonstrated the presence on the ermG mRNA of a 197 to 198-base leader. Within the leader are two small open reading frames (ORFs) capable of encoding 11- and 19-amino-acid peptides. Each ORF is preceded by a suitably spaced Shine-Dalgarno sequence. The ermG protein is encoded by a large ORF that encodes a 244-amino-acid protein, in agreement with the minicell results. This protein and the 19-amino-acid peptide are highly homologous to the equivalent products of ermC and ermA. We conclude, on the basis of this homology, that ermG encodes an rRNA transmethylase. The leader of ermG can be folded into a structure that sequesters the Shine-Dalgarno sequence and start codon for the large ORF (SD3). On the basis of these data and on the observed greater responsiveness of the ermG system than of the ermC system to low concentrations of erythromycin, we propose a model for the regulation of this gene in which the stalling of a ribosome under the influence of an inducer, while reading either peptide, suffices to uncover SD3 and allow translation of the rRNA transmethylase. The evolution of ermG is discussed.

Amino Acid Sequence↗

PubMed1987

Replication control genes of plasmid pE194.

pE194, a 3.7-kilobase plasmid, confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. The previously identified cop and repF genes of pE194 have been further localized by molecular cloning and mutational analysis together with DNA sequencing. The CfoIB fragment of pE194 is capable of autonomous replication and contains both genes. Most of this region has been resequenced, and two errors reported in a previous study have been corrected. The corrected sequence indicates that the replication region contains a single large open reading frame, which we propose encodes the repF product. Northern blot (RNA blot) analysis of this region detected six transcripts, all transcribed in the same direction as one another and opposite to repF. A 350-base transcript is synthesized from the region containing cop. No in vivo transcript for the repF gene was detected, but a protein was observed in an in vitro transcription-translation system which appears to be its product. An ochre mutation was inserted in the putative repF open reading frame, and a nonsense fragment was detected in the in vitro system. When carried passively on a pUB110 replicon, this mutant product appears capable of inhibiting pE194 replicons in trans. The pE194 origin of replication has been localized to within 200 bases.

Bacillus subtilis↗

PubMed1987

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.

Amino Acid Sequence↗

PubMed1987

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.

Bacillus subtilis↗

PubMed1987

Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis.

We isolated 28 mutants of Bacillus subtilis deficient in the development of competence by using the transposon Tn917lacZ as a mutagen. The mutant strains were poorly transformable with plasmid and chromosomal DNAs but were normally transducible and exhibited wild-type resistance to DNA-damaging agents. The mutations were genetically mapped, and the mutants were characterized with respect to their abilities to bind and take up radiolabeled DNA. All were defective in uptake, and some failed to bind significant amounts of DNA. The abilities of the mutant strains to resolve into two buoyant density classes on Renografin gradients were studied. Most resolved normally, but several banded in Renografin only at the buoyant density of noncompetent cells. The genetic mapping studies and the other analyses suggested that the mutations define a minimum of seven distinct com genes.

Bacillus subtilis↗