Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Long-circulating bacteriophage as antibacterial agents.

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

CTXφ immunity: Application in the development of cholera vaccines

CTXφ is a filamentous bacteriophage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae. CTXφ is unusual among filamentous phages because it encodes a repressor and forms lysogens. CTXφ can infect the existing live-attenuated V. cholerae vaccine strains derived from either the El Tor or classical V. cholerae biotypes and result in vaccine reversion to toxinogenicity. Intraintestinal CTXφ transduction assays were used to demonstrate that El Tor biotype strains of V. cholerae are immune to infection with the El Tor-derived CTXφ, whereas classical strains are not. The El Tor CTXφ repressor, RstR, was sufficient to render classical strains immune to infection with the El Tor CTXφ. The DNA sequences of the classical and El Tor CTXφ repressors and their presumed cognate operators are highly diverged, whereas the sequences that surround this “immunity” region are nearly identical. Transcriptional fusion studies revealed that the El Tor RstR mediated repression of an El Tor rstA-lacZ fusion but did not repress a classical rstA-lacZ fusion. Likewise, the classical RstR only repressed a classical rstA-lacZ fusion. Thus, similar to the mechanistic basis for heteroimmunity among lambdoid phages, the specificity of CTXφ immunity is based on the divergence of the sequences of repressors and their operators. Expression of the El Tor rstR in either El Tor or classical live-attenuated V. cholerae vaccine strains effectively protected these vaccines from CTXφ infection. Introduction of rstR into V. cholerae vaccine strains should enhance their biosafety.

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Cosmids from the 1A3–1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

rexB of bacteriophage λ is an anti-cell death gene

In Escherichia coli, programmed cell death is mediated through “addiction modules” consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of λrexB, one of the few genes expressed in the lysogenic state of bacteriophage λ, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3′,5′-bispyrophosphate (ppGpp) and thus by amino acid starvation. λRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of λRexB through its action on the ClpP proteolytic subunit. We also propose that the λrex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the “survival operon” of phage λ.

Plasticity in the neural coding of auditory space in the mammalian brain

Sound localization relies on the neural processing of monaural and binaural spatial cues that arise from the way sounds interact with the head and external ears. Neurophysiological studies of animals raised with abnormal sensory inputs show that the map of auditory space in the superior colliculus is shaped during development by both auditory and visual experience. An example of this plasticity is provided by monaural occlusion during infancy, which leads to compensatory changes in auditory spatial tuning that tend to preserve the alignment between the neural representations of visual and auditory space. Adaptive changes also take place in sound localization behavior, as demonstrated by the fact that ferrets raised and tested with one ear plugged learn to localize as accurately as control animals. In both cases, these adjustments may involve greater use of monaural spectral cues provided by the other ear. Although plasticity in the auditory space map seems to be restricted to development, adult ferrets show some recovery of sound localization behavior after long-term monaural occlusion. The capacity for behavioral adaptation is, however, task dependent, because auditory spatial acuity and binaural unmasking (a measure of the spatial contribution to the “cocktail party effect”) are permanently impaired by chronically plugging one ear, both in infancy but especially in adulthood. Experience-induced plasticity allows the neural circuitry underlying sound localization to be customized to individual characteristics, such as the size and shape of the head and ears, and to compensate for natural conductive hearing losses, including those associated with middle ear disease in infancy.

Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

Similarities and dissimilarities of phage genomes.

Genomic similarities and contrasts are investigated in a collection of 23 bacteriophages, including phages with temperate, lytic, and parasitic life histories, with varied sequence organizations and with different hosts and with different morphologies. Comparisons use relative abundances of di-, tri-, and tetranucleotides from entire genomes. We highlight several specific findings. (i) As previously shown for cellular genomes, each viral genome has a distinctive signature of short oligonucleotide abundances that pervade the entire genome and distinguish it from other genomes. (ii) The enteric temperate double-stranded (ds) phages, like enterobacteria, exhibit significantly high relative abundances of GpC = GC and significantly low values of TA, but no such extremes exist in ds lytic phages. (iii) The tetranucleotide CTAG is of statistically low relative abundance in most phages. (iv) The DAM methylase site GATC is of statistically low relative abundance in most phages, but not in P1. This difference may relate to controls on replication (e.g., actions of the host SeqA gene product) and to MutH cleavage potential of the Escherichia coli DAM mismatch repair system. (v) The enteric temperate dsDNA phages form a coherent group: they are relatively close to each other and to their bacteria] hosts in average differences of dinucleotide relative abundance values. By contrast, the lytic dsDNA phages do not form a coherent group. This difference may come about because the temperate phages acquire more sequence characteristics of the host because they use the host replication and repair machinery, whereas the analyzed lytic phages are replicated by their own machinery. (vi) The nonenteric temperate phages with mycoplasmal and mycobacterial hosts are relatively close to their respective hosts and relatively distant from any of the enteric hosts and from the other phages. (vii) The single-stranded RNA phages have dinucleotide relative abundance values closest to those for random sequences, presumably attributable to the mutation rates of RNA phages being much greater than those of DNA phages.