Genome and proteome analysis of phage E3 infecting the soil-borne actinomycete Rhodococcus equi.

We report on the characterization and genomic analysis of bacteriophage E3 isolated from soil and propagating in Rhodococcus equi strains. Phage E3 has a circular genome of 142 563 bp and is the first Myoviridae reported for the genus Rhodococcus and for a non-mycobacterial actinomycete. Phylogenetic analyses placed E3 in a distinct Myoviridae clade together with Mycobacterium phages Bxz1 and Myrna. The highly syntenic genomes of this myoviridal group comprise vertically evolving core phage modules flanked by hyperplastic regions specific to each phage and rich in horizontally acquired DNA. The hyperplastic regions contain numerous tRNA genes in the mycobacteriophages which are absent in E3, possibly reflecting bacterial host-specific translation-related phage fitness constraints associated with rate-limiting tRNAs. A structural proteome analysis identified 28 E3 polypeptides, including 15 not previously known to be virion-associated proteins. The E3 genome and comparative analysis provide insight into short-term genome evolution and adaptive plasticity in tailed phages from the environmental microbiome.

Starvation/stationary-phase survival of Rhodococcus erythropolis SQ1: a physiological and genetic analysis.

The adaptation of Rhodocccus erythropolis SQ1 to energy and carbon starvation was investigated in terms of both the capacity to survive starvation and the contribution of a nutrient-induced stationary phase to cross-protection to other types of environmental stress. It was found that R. erythropolis SQ1 survives for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1%) or low (0.1%) glucose. Furthermore, early stationary-phase R. erythropolis SQ1 grown in CDM 0.1% exhibited enhanced resistance to heat and oxidative stress compared with exponential-phase cells. A second objective of this study was to identify genetic elements involved in starvation/stationary-phase survival. A mutant bank of R. erythropolis SQ1 generated by random transposon insertion mutagenesis was screened; four mutants lost culturability when grown in CDM 1%. No drop in culturability was observed when these mutants were grown in CDM 0.1%. The DNA flanking transposon insertion could be recovered from three mutants. Transposon insertions were found in uvrB (UvrB, part of the DNA excision repair mechanism), between a putative guaB gene and another guaB-like gene, and between a gene encoding a putative phosphoglycerate mutase and putative thioredoxin/cytochrome c biogenesis genes. This represents a first study of the starvation/stationary-phase survival response of Rhodococcus, an organism of immense significance in environmental bioremediation and a number of industrial processes.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

DNA-binding activity of the Streptococcus thermophilus phage Sfi21 repressor.

The cloned Streptococcus thermophilus phage Sfi21 repressor open reading frame (orf) 127 gp protects a cell against superinfection with the homologous temperate, but not against virulent phages. As demonstrated by DNase protection assay and gel shift experiments, the repressor binds to a 25-bp operator site located upstream of the repressor gene. A second sequence-related operator was identified 265 bp apart at the 3'-end of orf 75, the topological equivalent of a cro repressor gene. The replacement of a bp at the middle or at the right side of the operator decreased substantially the affinity of the repressor for the operator. In gel shift assays, the 75 gp did not bind DNA from the genetic switch region. However, when increasing amounts of orf 75 gp containing cell extracts were added to orf 127 gp containing cell extracts, the repressor could no longer bind its operator site.

Transcription mapping as a tool in phage genomics: the case of the temperate Streptococcus thermophilus phage Sfi21.

For the lytic growth cycle of the temperate cos-site Streptococcus thermophilus phage Sfi21 a transcription map was developed on the basis of systematic Northern blot hybridizations. All deduced 5' ends were confirmed by primer extension analysis. Three time classes of transcripts were observed. Early transcripts were identified in four different genome regions. One prominent early mRNA of 4.8 kb length covered a group of 12 genes located between the origin of replication and the cos-site. Two short early mRNAs represented a single gene from the direct vicinity of the cos-site and the superinfection immunity gene from the lysogeny module, respectively. A fourth early transcript covered a group of four genes located between the lysin and the integrase gene. Middle transcripts of 2.1 and 5.8 kb length covered cro-like and ant-like repressor genes and the DNA replication module, respectively. Four types of late transcripts were identified. The transcripts covered the likely DNA packaging genes, the head morphogenesis module plus the major tail gene, the remainder of the tail genes, and the putative tail fiber plus lysis genes, respectively. Only the transcript from the head morphogenesis genes yielded defined late mRNA species. The transcription map concurred with most of the in silico predictions for the genome organization of phage Sfi21 except for the separation of the DNA replication module from a possible transcription regulation module. Most 5' ends of the transcripts determined in primer-extension experiments were not preceded by a consensus promoter sequence. The involvement of phage-encoded regulators for middle and late transcription was suggested by chloramphenicol-inhibition experiments.