Bacillus subtilis BY‐kinase PtkA controls enzyme activity and localization of its protein substrates

Bacillus subtilis BY‐kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP‐glucose dehydrogenases and single‐stranded DNA‐binding proteins. Our recent phosphoproteome study identified nine new tyrosine‐phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single‐stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA‐dependent phosphorylation. Because enzyme activity was not affected in other cases, we used fluorescent protein tags to study the impact of PtkA on localization of these proteins in vivo. For several substrates colocalization with PtkA was observed, and more importantly, the localization pattern of the proteins enolase, YjoA, YnfE, YvyG, Ugd and SsbA was dramatically altered in ΔptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.     

Plasmid replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH₂-Prothrombin-COOH $\text{Xa or thrombin}$ NH₂-Intermediate 3 + Intermediate 1-COOH; NH₂-Intermediate 1-COOH $\text{Xa}$ NH₂-Intermediate 4 + Intermediate 2-COOH; NH₂-Intermediate 2-COOH $\text{Xa}$ NH₂-A chain α-thrombin -S-S-B chain α-thrombin-COOH.Previous reports from this laboratory have demonstrated that the activation of prothrombin proceeds through several single-chain intermediates prior to the appearance of thrombin activity. (1) Subsequent studies have sequence of the prothrombin molecule can be deduced from the sequences of its activation intermediates and we are continuing our studies toward this goal.     

DNA replication during development of competence in Bacillus subtilis

Summary A new technique for studying DNA synthesis in competent cells of Bacillus subtilis has been developed.During competence development, the transformable cells are probably synthetizing DNA with a duplication time of approximately 90 min at 30° C; only when the maximum of competence is reached, does synthesis stop.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Bacillus subtilis mutant deficient in the major autolytic amidase and glucosaminidase is impaired in motility

Abstract The purified autolytic endo- β - N -acetylglucosaminidase of Bacillus subtilis AC327 was cleaved with cyanogen bromide, and the N-terminal amino acid sequence of one of the peptide fragments was determined. Then, a DNA fragment containing a part of the glucosaminidase gene was cloned into Escherichia coli JM109 using synthetic oligonucleotides as probes whose sequences had been deduced from the N-terminal amino acid sequence. Zymographic analysis showed that the resultant glucosaminidase-deficient strain lacked a 35-kDa lytic band in addition to a 90-kDa lytic one corresponding to the glucosaminidase. A double mutant strain deficient in the major two autolysins (amidase and glucosaminidase) exhibited greatly impaired motility on a swarm plate whereas the single mutant strains were motile.     

Genetic fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation.

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.     

Plasmid replication in DNA Ts mutants of Bacillus subtilis.

In an attempt to increase our understanding of plasmid replication in Bacillus subtilis we determined the effect of various dna Ts mutations [Gass, K. B., and Cozzarelli, N. R. (1973). J. Biol. Chem. 248, 7688–7700; Gross, J. D., Karamata, D., and Hempstead, P. G. (1968). Cold Spring Harbor Symp. Quant. Biol.33, 307–312; Karamata, D., and Gross, J. D. (1970). Mol. Gen. Genet.108, 277–287] on pUB110 replication. pUB110 is a kanamycin resistance plasmid originally isolated in Staphylococcus aureus and introduced into B. subtilis by transformation. At temperatures nonpermissive for chromosomal DNA synthesis dnaA13, dnaB19, dnaC6, dnaC30, dnaD23, dnaE20, and dnaI102 permit replication of the plasmid. In several cases this “amplification” continues until approximately equal amounts of plasmid and chromosomal DNA are present. dnaG34, dnaH151, dnaF133, mut-1, and polC26 affect both pUB110 and host DNA synthesis at nonpermissive temperatures. The last three mutations are known to affect the activity of DNA polymerase III (PolIII). When polC26 is incubated at a nonpermissive temperature, there is an accumulation of plasmid DNA with a density on EtBr-CsCl gradients intermediate between that of covalently closed circular (CCC) and open circular DNA. pUB110 can replicate in a strain which is deficient in DNA polymerase I (PolI). Finally, chloramphenicol (Cm) inhibits the replication of pUB110 as well as of chromosomal DNA.     

Identification and characterization of new DNA replication terminators in Bacillus subtilis

A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17-mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B. subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning <1O% of its length. Their DNA sequences are characterized by clearly identifiable A- and B-binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B. subtilis . Three of the four were tested for polarity in fork-arrest activity and exhibited the polarity expected. The total of six terminators now identified in B. subtilis have been named TerI-TerVI . TerI and TerII correspond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators ( TerIV ) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication-fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication-fork trap in each case.     

Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome

The Bacillus subtilis 168 dna-1 chromosome was labelled during sporulation with [3H]thymine for five minutes immediately before termination of replication. The isolated radioactive DNA was cleaved with BamHI (or SalI) and the resulting restriction fragments separated by agarose gel electrophoresis. The individual fragments, fractionated into a series of slices cut from the gel, were then cleaved with SalI (or BamHI) and the double-digest fragments identified by electrophoresis and fluorography. All major fragments and most minor ones present in a whole double-digest were assigned to BamHI and SalI parents. Such information enabled the construction of an unambiguous restriction map of 150 X 10(3) bases of the approximately 250 X 10(3) bases of DNA labelled in the five minutes. In conjunction with published data on the order of replication of restriction fragments as termination is approached, it was clear that most (105 X 10(3) bases) of the mapped DNA was replicated by a major fork moving in one direction towards a BamHI 24.8 X 10(3) base fragment. The 45 X 10(3) bases extending to the other side of this region were labelled only slightly, and presumably was replicated by a fork that approached the other in an opposite direction until its progress was blocked or severely impeded within this region at a site, referred to as terC, sometime (less than 5 min) earlier. The regions of the map replicated in the final 2.5 and 1.0 minute by the major fork were also identified.     

Isolation and partial characterization of Bacillus subtilis mutants impaired in DNA entry

Transformation-deficient mutants of Bacillus subtilis have been identified either by screening for a nuclease-deficient phenotype on methyl green-DNA agar or for nontransformability on transforming DNA-containing agar. After purification of the mutations causing a reduction in the entry of DNA, a set of isogenic entry-deficient strains was obtained. In addition to being entry deficient to various extents, the strains usually were less capable of association with DNA than the entry-proficient parent. Likewise, the specific transforming activity in the purified mutant strains continued to be less than that in the wild type. With the possible exception of one strain, no evidence was obtained that the mutant strains were impaired in recombination. Since the breakdown of transforming DNA to acid-soluble products correlated fairly well with the residual capacity of the strains to take up DNA, nucleolytic activity is likely to be involved in the entry of DNA in B. subtilis.     

Membrane attachment of the chromosome in Bacillus subtilis mutants temperature-sensitive in DNA replication

We have examined three mutants of Bacillussubtilis temperature sensitive in DNA initiation and one temperature sensitive in DNA elongation, in order to investigate whether these lesions can cause or can result in a detachment of the membrane-bound chromosomal region.Our results argue against any effect of the mutations examined on the association between the chromosome and the membrane.