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Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme Grant Agreement 646869.

The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5).

RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (, During his sabbatical stay in Cold Spring Harbor Laboratory, I.L. was supported by a “Salvador Madariaga” fellowship from the Spanish Ministry of Science and Innovation. A.T.-A. and J.V. were supported by Spanish Ministry of Science and Innovation “Ramon y Cajal” contracts. M.V. was supported by a Consejo Superior de Investigaciones Científicas JAE Predoctoral research contract. This work was supported by Spanish Ministry of Science and Innovation Grants BIO2008-05284-C02-01 and ERA-NET Pathogenomics PIM2010EPA-00606.

A surprising example of interspecies competition is the production by certain bacteria of hydrogen peroxide at concentrations that are lethal for others. A case in point is the displacement of Staphylococcus aureus by Streptococcus pneumoniae in the nasopharynx, which is of considerable clinical significance. How it is accomplished, however, has been a great mystery, because H 2 O 2 is a very well known disinfectant whose lethality is largely due to the production of hyperoxides through the abiological Fenton reaction. In this report, we have solved the mystery by showing that H 2 O 2 at the concentrations typically produced by pneumococci kills lysogenic but not nonlysogenic staphylococci by inducing the SOS response. The SOS response, a stress response to DNA damage, not only invokes DNA repair mechanisms but also induces resident prophages, and the resulting lysis is responsible for H 2 O 2 lethality. Because the vast majority of S. aureus strains are lysogenic, the production of H 2 O 2 is a very widely effective antistaphylococcal strategy. Pneumococci, however, which are also commonly lysogenic and undergo SOS induction in response to DNA-damaging agents such as mitomycin C, are not SOS-induced on exposure to H 2 O 2. This is apparently because they are resistant to the DNA-damaging effects of the Fenton reaction. The production of an SOS-inducing signal to activate prophages in neighboring organisms is thus a rather unique competitive strategy, which we suggest may be in widespread use for bacterial interference. However, this strategy has as a by-product the release of active phage, which can potentially spread mobile genetic elements carrying virulence genes.

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21 ) has been characterized by using Escherichia coli . This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP , produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli . Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP -overexpressing and aspP -deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli .