Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. smegmatis mutants.

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.

PthA4AT , a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance.

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT ) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to 'classical' TALEs.

Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria.

Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.