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GAS Genome Prophages

8.3.2.1 Prophages and Virulence Factors

The number and type of phages found in each GAS genome varies, as do the associated virulence genes. The insertion sites for the various phage genomes in the common GAS genome backbone is shown in Fig. 8.3, with several having the same insertion site. Although phage genomes are found throughout the bacterial chromosome, there is a prevalence of insertion near the terminus region on the lagging strand. Virulence genes are found associated with multiple attachment sites, and it is evident that frequent genetic rearrangements have occurred that have shuffled these virulence genes to different phage backgrounds.

The phage-associated virulence genes, while dispensable for phage replication and maturation, confer novel phenotypes upon the host streptococcus that increase its fitness. A considerable range of virulence genes are carried by the genomes of GAS prophages, including superantigens and streptodornases. A charac-

Fig. 8.3 Bacteriophage locations on the GAS backbone. The locations of the genome prophages on the S. pyogenes genome are shown on a generalized GAS DNA backbone based upon the M1 genome; prophages that share the same attachment site are boxed together.

Toxin genes that are associated with a particular prophage are indicated with the phage name. The rRNA operons are indicated as white blocks, while the cluster of virulence genes flanking emm are hatched. The origin of replication is indicated (oriC).

Fig. 8.3 Bacteriophage locations on the GAS backbone. The locations of the genome prophages on the S. pyogenes genome are shown on a generalized GAS DNA backbone based upon the M1 genome; prophages that share the same attachment site are boxed together.

Toxin genes that are associated with a particular prophage are indicated with the phage name. The rRNA operons are indicated as white blocks, while the cluster of virulence genes flanking emm are hatched. The origin of replication is indicated (oriC).

teristic of these genes is an atypically low G+C content relative to the rest of the genome, a feature shared with other genes of microbial pathogens transferred by horizontal gene transfer. For example, most of the pyrogenic exotoxins have a G+C content of about 30% whereas that of the genome is about 38.5%. While these atypical percentages of the virulence genes are suggestive of origination from an unknown ancestor, the actual genetic source for any of these ORFs is unknown and may also have originated within the species by gene duplication and descent. However, cryptic and sometimes expressed coding regions often are found in regions flanking the attachment sites in the genomes of temperate bacteriophages, even from those infecting nonpathogenic bacteria [81-83]. It seems possible that the origin of some virulence factors may have occurred by a process

Fig. 8.4 Phylogram of the S. pyogenes-identi-fied genome prophages and the related S. thermophilus temperate phages. The phylo-genetic tree analysis of the genome phages shows probable groups with related evolutionary histories. The prophages of GAS are indicated in black and the S. thermophilus phages in red. Multiple sequence alignment of the prophage genomes was done using ClustalX [86]. The genomes of S. thermophilus have been previously reported [85, 87-90].

Fig. 8.4 Phylogram of the S. pyogenes-identi-fied genome prophages and the related S. thermophilus temperate phages. The phylo-genetic tree analysis of the genome phages shows probable groups with related evolutionary histories. The prophages of GAS are indicated in black and the S. thermophilus phages in red. Multiple sequence alignment of the prophage genomes was done using ClustalX [86]. The genomes of S. thermophilus have been previously reported [85, 87-90].

of selection for function from preexisting phage genes. The relationships seen between some prophages of S. pyogenes and S. thermophilus (Fig. 8.4) raises the possibility that prototoxin genes may have arisen in a separate genera and reached GAS by horizontal transfer [84, 85].

Many of the genome prophages contain mutations or deletions that prevent the phage from entering the lytic cycle, thus fixing the prophage DNA in the strepto-coccal genome. The inactivation of a prophage while maintaining the associated virulence gene may be a frequent and evolutionarily favored event, preserving the benefit to the streptococcus conferred by the toxin while eliminating the danger of prophage induction and subsequent lysis. Frequent examples can be found in the GAS genome prophages where either a genetic defect (inactivation of the portal protein in phage SF370.2) or large genome deletion (MGAS10394.7) has fixed the associated toxin gene as a permanent element of the host. Homologous recombination with another phage genome could rescue the disabled prophage or recombine to generate a novel, functional phage. If the forces that lead to prophage decay are constant over time, then inactivated prophages may be long-term residents of their host, and conversely, the fully inducible phages may have been recently acquired. The number of inducible prophage genomes in the M3 MGAS315 strain may reflect this strain's status as a recently emerged clone [6]. The emergence of this highly virulent strain has been proposed to have resulted from the sequential addition of prophages (MGAS315.5, acquired in the 1920s; MGAS315.2, acquired around 1940; and MGAS315.4, acquired around 1980) to generate the current M3 clone [91]. The inducible status of these phages suggests that the timeframe for maintenance of intact prophages in GAS may be measured in decades. The M6 MGAS10394 strain has, by contrast, a large number of phage remnants, suggesting that the toxins associated with these prophages have been long-established host features.

8.3.2.2 Prophage Attachment Sites and Host Biology

Prophage integration occurs by a Campbell-type homologous exchange between a common sequence shared between the phage and host chromosomes (attP and attB, respectively). These duplications are of varying lengths, ranging from a few bases to nearly 100 bp and often represent coding regions of the host genome [92, 93]. In the S. pyogenes genome prophages, the identifiable duplications range from 12 bp in phage MGAS10394.1 to around 96 bp in T12 and the phages that share its attachment site [94]. In most bacterial species, the majority of duplications occur at the 3' end of some host target gene, and integration either leaves the target gene intact (via the duplication) or, in at least one case, provides an alternative carboxy terminus for the specified protein [92, 95]. In the S. pyogenes prophages, integration and duplication occurs frequently at either the 5' and 3' end of genes. Integration into the 5' end of a gene or its predicted promoter is an unusual strategy, potentially altering host gene function. Genes that are targeted for 5' integration in GAS include methyl-directed mismatch repair protein mutL, a dipeptidase gene, recombination protein recX, proB, a HAD-like hydrolase, and a conserved hypothetical protein. Other prophages appear to target the promoter region immediately upstream of the actual ORF, including the promoter regions of yesN, c-glu-tamyl kinase, and an iron-dependent repressor. Similarly, the transposon MGAS10394.4 with some prophage features separates the comE operon proteins 2 and 3, probably creating a polar mutation that silences protein 3. In all of these cases, the insertion of a prophage at the 5' end of a gene has the potential for altering host gene expression by introducing a polar mutation or an alternative promoter, and such transcription-altering prophages may be an important class of genetic regulatory elements in S. pyogenes in modifying the biology of their hosts.

8.3.2.3 Prophage Diversity

Pregenomic studies predicted that considerable diversity existed in GAS phages, with phages having a variety of toxin genes integrating into the bacterial chromosome at multiple sites [94, 96-98]. The GAS genome sequences have confirmed these predictions, and further, have revealed that virulence genes have often been reassorted with functional phage modules for integration and regulation or maturation and packaging to increase diversity. As shown in Fig. 8.3, the results of phage diversification have placed some virulence genes (such as speA) at multiple integration sites on the GAS chromosome. Thus, while natural selection has favored the preservation of overall phage gene order, the modules for integration, regulation, replication, DNA packaging, structural proteins, host lysis, and virulence have been often exchanged with alleles from other phages to generate the diversity that may well account for the sudden appearance of new strains of GAS.

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