The opportunistic human pathogen Pseudomonas aeruginosa regulates the production of numerous virulence factors via the action of two separate but coordinated quorum sensing systems, las and rhl. These systems control the transcription of genes in response to population density through the intercellular signals N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) and N-(butanoyl)-L-homoserine lactone (C(4)-HSL). A third P. aeruginosa signal, 2-heptyl-3-hydroxy-4-quinolone [Pseudomonas quinolone signal (PQS)], also plays a significant role in the transcription of multiple P. aeruginosa virulence genes. PQS is intertwined in the P. aeruginosa quorum sensing hierarchy with its production and bioactivity requiring the las and rhl quorum sensing systems, respectively. This report presents a preliminary transcriptional analysis of pqsA, the first gene of the recently discovered PQS biosynthetic gene cluster. We show that pqsA transcription required pqsR, a transcriptional activator protein encoded within the PQS biosynthetic gene cluster. It was also found that the transcription of pqsA and subsequent production of PQS was induced by the las quorum sensing system and repressed by the rhl quorum sensing system. In addition, PQS production was dependent on the ratio of 3-oxo-C(12)-HSL to C(4)-HSL, suggesting a regulatory balance between quorum sensing systems. These data are an important early step toward understanding the regulation of PQS synthesis and the role of PQS in P. aeruginosa intercellular signaling.

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV‐induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single‐stranded DNA‐binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV‐irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV‐induced DNA synthesis arrest may be mediated in part through phosphorylation‐related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.

Summary The sie2009 gene, which is situated between the genes encoding the repressor and integrase, on the lysogeny module of the temperate lactococcal bacteriophage Tuc2009, was shown to mediate a phage‐resistance phenotype in Lactococcus lactis against a number of bacteriophages. The Sie2009 protein is associated with the cell membrane and its expression leaves phage adsorption, transfection and plasmid transformation unaffected, but interferes with plasmid transduction, as well as phage replication. These observations indicate that this resistance is as a result of DNA injection blocking, thus representing a novel superinfection exclusion system. A polymerase chain reaction (PCR)‐based strategy was used to screen a number of lactococcal strains for the presence of other prophage‐encoded phage‐resistance systems. This screening resulted in the identification of two such systems, without homology to sie2009, which were shown to mediate a phage‐resistance phenotype similar to that conferred by sie2009. To our knowledge, this is the first description of a phage‐encoded superinfection exclusion/injection blocking mechanism in the genus Lactococcus.

Tuc2009 is a P335-type member of the tailed-phage supergroupLactic acid bacteria are economically important bacteria used in the production of fermented foods such as cheeses, yogurts, and sausages. Tuc2009 is a 38,347-bp lysogenic member of the P335 type of the Siphoviridae supergroup of noncontractile-tailed bacteriophages (GenBank accession no. NC_002703) and was originally identified in Lactococcus lactis subsp. cremoris UC509, a strain used in Cheddar cheese production, following mitomycin C induction (2, 42).Muralytic enzymes or lysins degrade the peptidoglycan (PG) matrix and play essential roles for both phages and bacteria. "Autolysins" is the term used for lysins which are produced by bacteria and involved in cell division, while the term "endolysins" refers to lytic enzymes involved in phage release. Some bacteria also produce lysins which act as class III bacteriocins. Lysins fall into three categories, glycosidases, amidases, and endopeptidases, depending on the type of chemical bond they cleave within the PG. Glycosidases can be further subdivided into the muramidases, glucosaminidases, and transglycosylases (55). Progeny release for many double-stranded-DNA-tailed phages has been shown to employ a lysis system involving one or more holins in conjunction with an endolysin. The holins function by forming pores in the cytoplasmic membrane of the host, thereby abolishing membrane potential and allowing the endolysin to access the PG layer.Lysins exhibit a modular design (16). While a portion (usually the N-terminal part in the case of endolysins) encodes bond cleavage, a second segment is involved in substrate binding. This is believed to help the enzymatic efficiency and specificity of such muralytic enzymes by locating the active motif directly at the site of the substrate and causing endolysins to lyse only those bacteria possessing both the specifically recognized binding region and the target bond of the cleaving domain. It is this specificity of target recognition that could make lysins attractive therapeutic agents. Indeed, studies have demonstrated the usefulness of lysins by specifically lysing streptococci which had colonized mice (38). The lysin is thus said to demonstrate independently functioning domains, as shown for the choline-binding motif of the majority of lysins of Streptococcus pneumoniae and its phages (16) and the endolysin of Tuc2009 (50). Furthermore, the level of homology between these modules from endolysins and autolysins is supportive of the modular theory of phage evolution, as it indicates that the genes encoding such enzymes have arisen as a result of genomic exchange and rearrangement (16).While the cellular PG layer gives structural support to the bacterium, it also represents a formidable barrier across which the phage must transport its DNA during the infection process. Several proteins used by phages infecting gram-negative bacteria to perform this task of "hole punching" have been characterized (45). Phages T4, T7, PRD1, and 6, all of which infect gram-negative ho...

Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep 2009 and orf17. One of these genes, rep 2009 , codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori 2009 ), which was identical for all four phages. DNA fragments representing the ori 2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori 2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep 2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case.