A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain.

A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains.

Mode of action of linenscin OC2 against Listeria innocua.

Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria. It is also able to lyse eucaryotic cells. The mode of action of linenscin OC2 on the Listeria innocua cytoplasmic membrane and the effects of environmental parameters were investigated. Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state. Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption. Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane. We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.

Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2.

Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli.

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.

Overreplication of the origin region in the dnaB37 mutant of Bacillus subtilis: postinitiation control of chromosomal replication.

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after accumulation of initiation proteins at 45 degrees C, we have shown, by extensive DNA.DNA hybridization analysis, that the origin region is replicated in excess (approximately 2-fold). However, this replication is limited to a region of about 120-175 kilobases on either side of the origin. This has been confirmed by autoradiographic analysis of the overreplicated region. During the second round of synchronized replication at 30 degrees C, replication in fact appears to resume from the stalled forks on either side of the origin. We propose that in B. subtilis, in addition to a first level of control at the origin, a second level of control exists downstream of the origin in order to limit overreplication of the chromosome. These two controls might normally be tightly coupled. We suggest that the second level of control is exerted through the reversible inhibition of replisome movement at specific regions on either side of the origin.

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins.

When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45 degrees C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4 kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.

An RNA-DNA copolymer whose synthesis is correlated with the transcriptional requirement for chromosomal initiation in Bacillus subtilis contains ribosomal RNA sequences.

During synchronous replication induced in a temperature-sensitive initiation mutant of Bacillus subtilis, we previously isolated an RNA covalently linked to DNA. This molecule was synthesized at specific times, correlated with the period of transcription that is required to initiate a new round of replication. In this paper, we show that both RNA and DNA components of the RNA-DNA molecule hybridized with the coding strand for ribosomal RNA. Competition hybridization experiments also demonstrated that ribosomal RNA sequences represent the great majority of the RNA that is linked to DNA. Both RNA and DNA components of the RNA-DNA molecule also hybridized with a region close to the origin of replication, in particular with E19 and E22, two restriction fragments that are replicated early. These fragments in fact form part of the ribosomal operon rrnO. The role of the RNA-linked DNA molecule in initiation in B. subtilis and the possibility that this molecule emanates directly from rrnO rather than from other ribosomal RNA genes are discussed.

Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication.

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.

Isoaccepting species of serine tRNA coded by bacteriophage T5sto.

By aminoacyl-tRNA-DNA hybridization and chromatographic analysis, evidence was provided that the bacteriophage T5stO codes for two tRNAser species. Trinucleotide- or polynucleotide-stimulated binding experiments assigned the codons UCC or UCU to these two tRNAser species. They also suggested that the synthesis of these two tRNAser species does not modify the reading capacity for codons less used in Escherichia coli F and corresponds to a different situation compared with the T4-coded tRNA's.