Nucleotide sequence of the operators of lambda ultravirulent mutants.

The nucleotide sequence of the operators of ultravirulent mutants of lambda, able to grow on host cells with elevated repressor levels, was determined. It appears that ultravirulence in lambda requires multiple mutational events at the operator sequences. OL1, OL2, and OL3 operator sites are the target of mutational changes in ultravirulent phages indicating that these sites participate in vivo in repression of the PL promoter. No changes were found in the OR3 sequence, in contrast there is a mutation in OR2 and two mutations in OR1, in both lambda 668 and lambda 2668 phages. This mutated operator structure accounts for the constitutive expression of their PR promoter either in cells overproducing the lambda repressor or in cells overproducing the cro gene product. A model of the structure of the lambda operator site is proposed. The nucleotide sequence in each site can be divided into two functionally different subsets, one of which is recognized by the repressor while the other stabilizes the repressor-operator interaction.     

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Identification of related genes in phages phi 80 and P22 whose products are inhibitory for phage growth in Escherichia coli IHF mutants.

Bacteriophage lambda grows in both IHF+ and IHF- host strains, but the lambdoid phage phi 80 and hybrid phage lambda (QSRrha+)80 fail to grow in IHF- host strains. We have identified a gene, rha, in the phi80 region of the lambda(QSRrha+)80 genome whose product, Rha, inhibits phage growth in an IHF- host. A search of the GenBank database identified a homolog of rha, ORF201, a previously identified gene in phage P22, which similarly inhibits phage growth in IHF- hosts. Both rha and ORF201 contain two possible translation start sites and two IHF binding site consensus sequences flanking the translation start sites. Mutations allowing lambda (QSRrha+)80 and P22 to grow in IHF- hosts map in rha and ORF201, respectively. We present evidence suggesting that, in an IHF+ host, lambda(QSRrha+)80 expresses Rha only late in infection but in an IHF- host the phage expresses Rha at low levels early in infection and at levels higher than those in an IHF+ host late in infection. We suspect that the deregulation of rha expression and, by analogy, ORF201 expression, is responsible for the failure of phi80, lambda(QSRrha+)80, and P22 to grow in IHF mutants.     

A universal instinct for designing thermoregulated promotors in gram-positive bacteria]

The construction of plasmid pVKH300, which is useful for modifying any promoter into the thermoregulated form in B. subtilis cells, is presented. The main features of the plasmid are the presence of effectively expressed in B. subtilis lambda C1857 gene and recognition site of BglII restriction enzyme between OR2 and OR3 lambda phage operator sites. Promoterless alpha-amylase gene of B. amyloliquefaciens is used as a reporter gene for promoter cloning into BglII site of pVKH300. Examples of promoter-containing DNA fragments cloning with pVKH300 as vector are presented. It was found that the best regulated promoter, in a plasmid named pVKH332, was cloned in such a way that the distance between central nucleotides of OR2 and OR3 is equal to integer number of DNA helix turns (84 b.p. in the case).     

A novel antivirulence element in the temperate bacteriophage HK022.

Lysogens of the temperate lambdoid phage HK022 are immune to superinfection by HK022. Superinfection immunity is conferred in part by the action of the HK022 CI repressor at the O.R operators. In this work, we have identified an additional regulatory element involved in immunity. This site, termed OFR (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from OR. The behavior of phage containing a mutation in OFR suggests that the wild-type site functions as an antivirulence element. HK022 OFR- mutants were able to form turbid plaques indistinguishable from those of the wild type. However, they gave rise to virulent derivatives at a far higher frequency than the wild type (approximately 10(-5) for OFR- versus about 10(-9) for the wild type). This frequency was so high that cultures of HK022 OFR- lysogens were rapidly overgrown by virulent derivatives. Whereas virulent mutants arising from a wild-type OFR+ background contained mutations in both OR1 and OR2, virulent derivatives of the OFR- mutant phage contained a single mutation in either OR1 or OR2. We conclude that the wild-type OFR site functions to prevent single mutations in OR from conferring virulence. The mechanism by which OFR acts is not yet clear. Both CI and Cro bound to OFR and repressed a very weak rightward promoter (PFR). It is unlikely that repression of PFR by CI or Cro binding to OFR can account in full for the antivirulence phenotype conferred by this element, since PFR is such a weak promoter. Other models for the possible action of OFR are discussed.     

Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda

Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage λ are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two λ promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.