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The genes encoding the lysis proteins of Lactococcus lactis bacteriophage phi LC3 were cloned, sequenced, and expressed in Escherichia coli. The phi LC3 lysis genes, lysA and lysB, encode a membrane-disrupting protein (LysA) of 88 amino acids, and a cell wall degrading protein (LysB) of 429 amino acids, which shares significant sequence similarity with lysins from the Streptococcus pneumoniae phages Cp-1, Cp-7, and Cp-9, and Lactobacillus delbrueckii phage mv1. Both LysA and LysB function in E. coli, as judged by lysis of the E. coli host cells and by lytic activity against lactococcal cells when the cloned lysA and lysB genes are expressed. The LysA protein possesses two putative transmembrane helices and highly charged N- and C-termini, and is structurally similar to phage holins that are known to induce lesions in the inner membrane through which phage endolysin can be released to its cell wall substrate. The C-terminal end of LysB contains two highly homologous sequence repeats of 43 amino acids. The LysB repeats show strong sequence similarity to repeats found in lytic enzymes from other Gram-positive bacteria and from Bacillus subtilis phage phi 29 and PZA, as well as in some functionally unrelated proteins, and they are possibly involved in binding of the enzyme to the cell wall substrate. The organization of the dual phi LC3 lysis system supports earlier suggestions that exchange of modular units is an important principle in protein evolution.

Plasmids carrying the cohesive end region from temperate lactococcal bacteriophage PhiLC3 could be packaged in vivo by PhiLC3 and transduced into its host strain, Lactococcus lactis subsp. cremoris NCDO 1201. The transduction frequencies were between 10 and 10 transducing particles per PFU, depending on the size of the phage DNA insert. This transduction system is limited to only certain lactococcal strains. The PhiLC3 cohesive site region (cos) appears to play an important role in plasmid transduction.

The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The phi LC3 phage attachment site, attP, was mapped and sequenced. DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. The attB core sequence is located within the C-terminal part of an open reading frame of unknown function. The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP. The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family. Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed.

The nucleotide sequence of the DNA regions flanking the cohesive end site of the temperate lactococcal bacteriophage phi LC3 was determined. Four pairs of sequence repeats of 8 to 13 base-pairs in length were revealed close to the cohesive ends. These repeats possibly represent signal sequences involved in phage DNA maturation and packaging. The start of an open reading frame was localized only 42 nucleotides from the left end of the phi LC3 phage double-stranded region, implying that transcription probably proceeds across the cos site. Compilation of the characteristics of the DNA termini of bacteriophages with complementary cohesive DNA ends suggests that phages of Gram-negative bacteria generally carry 5'-protruding single-strands whereas those of Gram-positive hosts carry 3'-protruding ends.

The bacteriophage P2 ogr gene encodes an essential 72-amino-acid protein which acts as a positive regulator of P2 late transcription. A P2 ogr deletion phage, which depends on the supply of Ogr protein in trans for lytic growth on Escherichia coli C, has previously been constructed. E. coli B and K-12 were found to support the growth of the ogr-defective P2 phage because of the presence of functional ogr genes located in cryptic P2-like prophages in these strains. The cryptic ogr genes were cloned and sequenced. Compared with the P2 wild-type ogr gene, the ogr genes in the B and K-12 strains are conserved, containing mostly silent base substitutions. One of the base substitutions in the K-12 ogr gene results in replacement of an alanine with valine at position 57 in the Ogr protein but does not seem to affect the function of Ogr as a transcriptional activator. The cryptic ogr genes are constitutively transcribed, apparently at a higher level than the wild-type ogr gene in a P2 lysogen.

The temperate bacteriophage phiLC3, isolated from Lactococcus lactis subsp. cremoris, has an isometric head and a flexible tail containing 1 major protein and 8 minor proteins. Infection of a permissive L. lactis host strain yields a burst of about 50 phages per infected cell with a latent period of 60 min. A detailed restriction map of the phage chromosome was constructed by using 12 different restriction enzymes. The phage chromosome is a 33-kb linear double-stranded DNA molecule with unique cohesive ends and with a G + C content of 36.5%. Chemical sequencing of the DNA ends revealed 13-base 3' extended complementary single strands with a relatively high percentage of G + C. Pulsed-field gel electrophoretic analysis of DNA from a strain lysogenized with phiLC3 was used to localize the prophage to a 320-kb BamHI restriction endonuclease fragment from the host chromosomal DNA. This result indicates that lysogeny involves integration of the phage into the host chromosome. A spontaneous phiLC3 clear plaque mutant that was unable to give rise to lysogens was isolated.

The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an S1 mapping assay. During a normal P2 infection, ogr gene transcription starts from Pogr at an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pogr but is unable to transcribe the late genes. A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.

We have fused the promoter (PF) for the P2 late FETUD operon to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, delta. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpoA109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of PF were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides -69 and -64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.

The phenotypes and genotypes of 26 beta lactamase (penicillinase) producing strains of Neisseria gonorrhoeae (PPNG) from African countries were investigated. Using the restriction enzyme technique nine different restriction enzyme patterns were found, two of them in 15 strains. Of the 26 strains, 16 belonged to serogroup WI (containing protein type IA) and 10 to serogroup WII/III (containing protein IB). Among the IA strains four different serovars were represented, whereas six serovars were found among the IB strains. Five different auxotypes were identified, of which proline requiring (found in 12 strains) and prototrophic (found in 10 strains) dominated. Twelve strains harboured a 4.4 megadalton as well as a 24.5 megadalton plasmid. A 3.2 megadalton plasmid was found in 14 strains, one of which also harboured a 24.5 megadalton plasmid. The 2.8 megadalton cryptic plasmid was present in all 26 strains. The MICs of doxycycline ranged from 0.25 to 2.0 mg/l; the MIC 50% for WI strains was 0.25 mg/l and for WII/WIII strains 1.0 mg/l. A total of 10 different combinations of restriction enzyme pattern, serovar, auxotype, and plasmid were seen in the 16 WI strains compared with eight such combinations in the 10 WII/WIII strains. As expected, the restriction enzyme technique and serological classification gave better differentiation than plasmid profiles and susceptibility to doxycycline. More relevantly, however, these techniques also compared favourably with auxotyping. When the different systems were combined, the sensitivity was greatly increased.

The bacteriophage P2 late control gene ogr was cloned and precisely localized by deletion analysis in vitro. The DNA sequence of the ogr gene containing the ogr1 mutation was determined. The sequence translates into a basic protein of a molecular weight of 8300. Plasmids overproducing the ogr gene product were constructed, and the ogr gene product was identified by polyacrylamide gel electrophoresis.

Lipopolysaccharides (LPS) from Leptotrichia buccalis, strains L11, ATCC 14201 and ATCC 19616 were partially degraded with 1% acetic acid. The acid soluble polysaccharides were subjected to gel filtration. These experiments showed that the carbohydrate moiety of LPS from ATCC 14201 and ATCC 19616 were similar, consisting of a core oligosaccharide unsubstituted by 0-side chains containing galactose, glucose and D-glycero-D-manno-heptose as neutral sugars. The core analogous fraction isolated from L11 LPS contained the same three neutral sugars, but in different ratios.

Lipopolysaccharide (LPS) was extracted with aqueous phenol from Leptotrichia buccalis strains L 11, ATCC 14201 and ATCC 19616. Virtually all the LPS was found in the water phase. LPS could also be extracted with phenol/chloroform/petroleum ether, but not with cold trichloroacetic acid. All LPS preparations contained D-glycero-D-manno-heptose, galactose, glucose, glucosamin, n-dodecanoate, 3-hydroxy-n-tetradecanoate and phosphorus. In addition LPS from L11 contained 2-keto-3-deoxyoctonate and an unidentified aldose. LPS from L11 was serologically distinct from LPS of ATCC 14201 and ATCC 19616. LPS from the two latter strains crossreacted.

Uptake of glutamate into vesicles of Halobacterium salinarium has been studied during respiration and in the nonrespiring state. Uptake requires respiration or a minimum gradient in NaCl, which is consistent with an Na+ symport mechanism for uptake, as proposed for H. halobium. By replacing KCl or NaCl by choline chloride, it has been possible to distinguish between the effects of gradients and/or absolute concentration effects of NaCl and KCl. Uptake depends on the concentration of KCl on the inside, but not on a gradient in KCl. This points to a role for K+ as a regulator of uptake rate, but not of total uptake. The uptake of glutamate is not inhibited by a number of acids with similar chemical groups. Inhibition is, however, caused by D-glutamate. This indicates a specific transport site for glutamate. Parallel results are obtained for binding of glutamate to a Triton extract of the vesicle membrane. The variation in binding and uptake properties with the salt concentration is discussed with reference to transport kinetics.

The ogr gene of bacteriophage P2 codes for a basic protein of 72 amino acids which is thought to be essential for activation of P2 late gene transcription. However, conditionally lethal mutations in the ogr gene have never been isolated. We have constructed a P2 ogr deletion mutant by in vitro techniques. This deletion phage, P2-del15, grows in a host which provides the ogr gene product in trans from a plasmid but fails to grow in hosts lacking the ogr plasmid. This demonstrates that the ogr gene is essential for P2 lytic growth. The deletion in P2del15 has removed about half of the carboxy-terminal part of the ogr gene. The transcript from this deletion mutant can be distinguished from the wild-type transcript by S1 nuclease protection. The analysis of such transcripts suggests that the ogr gene product may negatively regulate its own transcription.