Biomedical subjects
Publications and source records attributed to D H Bechhofer.
The ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post-transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.
Two genetic determinants are sufficient for small derivatives of broad host-range plasmid RK2 to replicate in different Gram-negative bacteria: trfA, which encodes a replication initiator, and oriV, the origin of replication. In this study, nonessential RK2 determinants in the region encoding oriT, the origin of conjugative transfer, and the korA-korB operon, whose products regulate trfA expression, were tested for their effects on the stability of mini-RK2 plasmids in eight different hosts. We found that determinants of both regions can substantially alter plasmid stability, but the effects are not uniform in all hosts. The results also indicate that the effects of the korA-korB operon extend beyond that of the regulation of trfA transcription. This study further illustrates the different requirements for stable plasmid maintenance in diverse bacteria and the ability of wild-type RK2 to adapt to a variety of intracellular environments. The data also provide further evidence for the involvement of different regions of RK2 for stable maintenance in various hosts.
In Bacillus subtilis, the ermC gene encodes an mRNA that is unusually stable (40-min half-life) in the presence of erythromycin, an inducer of ermC gene expression. A requirement for this induced mRNA stability is a ribosome stalled in the ermC leader region. This property of ermC mRNA was used to study the decay of mRNA in B. subtilis. Using constructs in which the ribosome stall site was internal rather than at the 5' end of the message, we show that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site. Our results indicate that ermC mRNA is degraded by a ribonucleolytic activity that begins at the 5' end and degrades the message in a 5'-to-3' direction.
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.
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.
The korB gene of broad host-range plasmid RK2 prevents host-cell lethality by kilB and negatively controls RK2 replication. We precisely mapped the limits of korB to a region near korA, an autoregulated gene involved in control of several RK2 genes. The following results show that korA and korB are cotranscribed from the korA promoter: Mutants deleted for the korA promoter fail to express korB, even with korA function supplied in trans; the korA promoter is nonessential to korB if a heterologous promoter is present; and RNA produced in vivo has both korA- and korB-specific sequences. Analysis of polypeptides synthesized from wild-type and mutant korB plasmids in maxicells revealed that korB encodes a 52-kDa polypeptide, whose activity is extremely sensitive to changes in its carboxyl terminus but relatively unaffected by replacement of its amino terminus. The minimal korB-encoding region allowed us to identify two new regulatory functions, both of which duplicate previously known functions of korA. First, korB alone was found to control the kilB1 component of kilB, thus resolving the paradox of korA-independent control of kilB. Second, analysis of polypeptides from the korA-korB region in the presence and absence of korB, and studies with the korA promoter fused to the chloramphenicol acetyltransferase structural gene (cat) showed that korB, like korA, autoregulates expression of the korA-korB operon. We suggest that korA and korB gene products act as co-repressors in the control of certain RK2 genes.
ermC specifies an rRNA methyltransferase that confers resistance to erythromycin. The expression of this determinant is induced by the addition of erythromycin. The induction mechanism has been shown to operate posttranscriptionally, and its mechanism has been elucidated. We now show that synthesis of the ermC gene product in Bacillus subtilis is also autoregulated by a mechanism operating on the level of translation. The synthesis of methyltransferase was shown to be gene dosage compensated by Western blot analysis. Several mutants were analyzed that specify altered ermC gene products and are deregulated. Analysis of mutants and of the wild-type strain by Northern blotting demonstrated that autoregulation is posttranscriptional. We suggest a translational repression model in which the ermC methyltransferase binds to its own mRNA, at a region that resembles the methylation target site on 23S rRNA. The overall control of ermC expression is discussed in light of these multiple regulatory mechanisms.
The kil and kor genes of RK2 are novel genetic determinants further that the kil and kor network constitutes a replication regulon, and that perhaps the function of this regulon is to ensure expression of trfA at appropriate levels. The complexity of this regulon may reflect an ability of the system to adapt to the intracellular environments of a variety of hosts. Indeed, there is tantalizing evidence that regions encoding kil or kor genes are important to host range (1,2,6,28; Schmidhauser and Helinski, pers. comm.). We are therefore hopeful that the study of these genes and the eventual determination of the molecular basis of their actions will lead to a complete understanding of the replication control and broad host range capability of IncP plasmids.
We previously reported that broad-host-range plasmid RK2 encodes multiple host-lethal kil determinants (kilA, kilB1, kilB2, and kilC) which are controlled by RK2-specified kor functions (korA, korB, and korC). Here we show that kil and kor determinants have significant effects on RK2 replication control. First, korA and korB inhibit the replication of certain RK2 derivatives, unless plasmid replication is made independent of the essential RK2 gene trfA. Second, kilB1 exerts a strong effect on this interaction. If the target plasmid is defective in kilB1, sensitivity to korA and korB is enhanced at least 100-fold. Thus, korA and korB act negatively on RK2 replication, whereas kilB1 acts in a positive manner to counteract this effect. A mutant RK2 derivative, resistant to korA and korB, was found to have fused a new promoter to trfA, indicating that the targets for korA and korB are at the 5' end of the trfA gene. We constructed a trfA-lacZ fusion and found that synthesis of beta-galactosidase is inhibited by korA and korB. Thus korA, korB, and kilB1 influence RK2 replication by regulating trfA expression. We conclude that the network of kil and kor determinants is part of a replication control system for RK2.
Plasmid pRP761 is a derivative of the promiscuous plasmid RP4, which has a Tn76 insert 1.8 kb from its EcoRI site within the trfB region (Barth 1979). This mutation was pleiotropic, having three effects: the plasmid is unstably maintained in E. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. We show that pRP761 has reduced expression from both its korA and korB genes and that Tn76 has inserted between them. Fragment exchange experiments showed that this is the only mutant region in pRP761 and is therefore solely responsible for the pleiotropic effects. A spontaneous deletion derivative pRP761-6 has lost Tn76 and its adjacent kilA and korA genes: it has reacquired stability, does not inhibit host growth but is still reduced in its host-range. The provision of cloned korA+ in trans complements the first two phenotypic effects in pRP761 to a large extent, but neither korA+ alone nor korA+ with korB+ complements the host-range reduction in pRP761 or pRP761-6. A possible explanation for these results is that there is a site between korA and korB, affected by the Tn76 insert, that is essential to stable replication of these plasmids in some of their bacterial hosts.
The broad-host-range plasmid RK2 encodes three host-lethal kil genes whose actions are controlled by specific kor genes. We have shown previously that the 0' to 5.5' region of RK2 encodes both kilA and korC. Because of the lethal effect of kilA, plasmids with this region cannot be maintained in Escherichia coli unless the RK2 korA gene is also present. To investigate korC in the absence of kilA and therefore of korA, we first mapped kilA and korC to specific segments of the cloned 0' to 5.5' region. This allowed us to construct a korC+ plasmid missing the kilA region and thereby removed the need to have korA in the cell. We found that this korC-encoding plasmid alone is insufficient to control kilC. The korA function is required, and it can be supplied in trans. We also constructed a kilA+ korC- plasmid and found that korA is sufficient to control kilA. Thus, in addition to acting negatively to control kilA, korA acts positively to allow korC control of kilC. This korA dependence of korC is bypassed in a rho-115 mutant of E. coli. We consider the possibility that korA product acts as an antiterminator of transcription in korC expression.
From our earlier work, we know that the korA gene of broad host range plasmid RK2 is located within the 50.4'-56.4' region. By additional subcloning of this region, we have mapped korA to the segment between the HaeII site at 55.0' and the HincII site at 55.6'. The direction of korA transcription (55.6' to 55.1') was determined by two methods: (1) inactivation of korA expression signals and fusion of the structural gene to other promoters; and (2) hybridization analysis of korA-specific RNA's synthesized in vivo. We have determined the nucleotide sequence of the korA region. A potentially strong promoter overlaps the HincII site at 55.6', and there is a coding region which specifies the putative korA polypeptide. That this is the korA gene was supported by sequence analysis of Ba131-generated deletion mutants of korA. The sequence shows the korA product to be a small, basic polypeptide of 101 amino acids.
Cloning of specific regions of RK2, a broad host range incompatibility group P plasmid, has revealed three genes: kilA, kilB, and kilC. Each of these genes can cause loss of viability of an Escherichia coli host. This effect on the host is normally prevented by the functions of three additional RK2 genes: korA, korB, and korC. Each kor gene is specific for a particular kil gene. The kil and kor genes are located in four distinct regions of the RK2 genome. The three kil genes are not clustered and, with the possible exception of kilA, they are also well separated from their corresponding kor genes. We have found that the korA and korB determinants are not peculiar to RK2 but instead are highly conserved throughout the incompatibility group P plasmids.