Cross-species communication via agr controls phage susceptibility in Staphylococcus aureus.

Bacteria use quorum sensing (QS) to coordinate group behavior in response to cell density, and some bacterial viruses (phages) also respond to QS. In Staphylococcus aureus, the agr-encoded QS system relies on accumulation of auto-inducing cyclic peptides (AIPs). Other staphylococci also produce AIPs of which many inhibit S. aureus agr. We show that agr induction reduces expression of tarM, encoding a glycosyltransferase responsible for α-N-acetylglucosamine modification of the major S. aureus phage receptor, the wall teichoic acids. This allows lytic phage Stab20 and related phages to infect and kill S. aureus. However, in mixed communities, producers of inhibitory AIPs like S. haemolyticus, S. caprae, and S. pseudintermedius inhibit S. aureus agr, thereby impeding phage infection. Our results demonstrate that cross-species interactions dramatically impact phage susceptibility. These interactions likely influence microbial ecology and impact the efficacy of phages in medical and biotechnological applications such as phage therapy.

An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCCmec Cassette.

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-Cas; however, in the human pathogen Staphylococcus aureus, CRISPR-Cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-Cas in genomes of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Denmark. Only 2.9% of the strains carried CRISPR-Cas systems, but for strains of sequence type ST630, over half were positive. All CRISPR-Cas loci were type III-A and located within the staphylococcal cassette chromosome mec (SCCmec) type V(5C2&5), conferring ß-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-Cas positive strains, and almost identical SCCmec cassettes, CRISPR arrays, and cas genes are present in staphylococcal species other than S. aureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmec cassette containing CRISPR-Cas is excised from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage phiIPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or circumvented by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests that native S. aureus CRISPR-Cas offers only partial immunity and in nature may work in tandem with other defense systems. IMPORTANCE CRISPR-Cas is an adaptive immune system protecting bacteria and archaea against mobile genetic elements such as phages. In strains of Staphylococcus aureus, CRISPR-Cas is rare, but when present, it is located within the SCCmec element, which encodes resistance to methicillin and other ß-lactam antibiotics. We show that the element is excisable, suggesting that the CRISPR-Cas locus is transferable. In support of this, we found almost identical CRISPR-Cas-carrying SCCmec elements in different species of non-S. aureus staphylococci, indicating that the system is mobile but only rarely acquires new spacers in S. aureus. Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages that can overload the system or form escape mutants. Thus, we propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems and so may work with other defense systems to prevent phage-mediated killing.

Screening for Highly Transduced Genes in Staphylococcus aureus Revealed Both Lateral and Specialized Transduction.

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds, we identified a pattern of transduction that encompassed mobile genetic elements as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirmed previous reports of lateral transduction of bacterial DNA downstream of the integrated phage but also indicated a novel form of specialized transduction of DNA upstream of the phage. These findings illustrated the complexity of transduction processes and increased our understanding of the mechanisms by which phages transfer bacterial DNA. IMPORTANCE Horizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses play a major role in facilitating the horizontal transfer. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We showed that the genomic location of bacterial DNA relative to where bacteriophages integrated into that bacterial genome affected how frequently that DNA was transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrated both lateral and specialized transduction. The use of mutant libraries to investigate the genomic patterns of bacterial DNA transferred between cells could help us understand how horizontal transfer influences virulence and resistance development.