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Enterococcus faecalis is often coisolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their coprevalence, the polymicrobial interactions of these two species in biofilms and under iron-restricted conditions remain unexplored. Here, we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase ( ldh1 ) gives rise to l-lactate production during fermentative growth. We find that an E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced under iron-restricted conditions, resulting in increased lactic acid exported and, consequently, a reduction in local environmental pH. Together, our results suggest that E. faecalis synergistically inhibits P. aeruginosa growth by decreasing environmental pH and l-lactate-mediated iron chelation. Overall, this study emphasizes the importance of the microenvironment in polymicrobial interactions and how manipulating the microenvironment can impact the growth trajectory of bacterial communities. IMPORTANCE Many infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored under iron-restricted conditions. Here, we explore the polymicrobial interactions between commonly coisolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron and also lowers the environmental pH to antagonize P. aeruginosa growth under iron-restricted conditions. Our findings provide insights into polymicrobial interactions between bacteria and how manipulating the microenvironment can be taken advantage of to better control infections.

Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes. IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay., (Copyright © 2019 Matysik and Kline.)

Like many bacteria, Enterococcus faecalis encodes a number of adhesins involved in colonization or infection of different niches. Two well-studied E. faecalis adhesins, aggregation substance (AS) and endocarditis- and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting that they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here, we examined if they are coexpressed on the same cells and what is the functional impact of coexpression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation. IMPORTANCE Most bacteria express multiple adhesins that contribute to surface attachment and colonization. However, the network and relationships between the various adhesins of a single bacterial species are less well understood. Here, we examined two well-characterized adhesins in Enterococcus faecalis , aggregation substance and endocarditis- and biofilm-associated pili, and found that they exhibit distinct functional contributions depending on the growth stage of the bacterial community. Pili interfere with aggregation substance-mediated clumping and plasmid transfer under planktonic conditions, whereas the two adhesins structurally complement one another during biofilm development. This study advances our understanding of how E. faecalis , a ubiquitous member of the human gut microbiome and an opportunistic pathogen, uses multiple surface structures to evolve and thrive., (Copyright © 2018 Afonina et al.)

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Gene conversion mediates the variation of virulence-associated surface structures on pathogenic microorganisms, which prevents host humoral immune responses from being effective. One of the best-studied gene conversion systems is antigenic variation (Av) of the pilin subunit of the Neisseria gonorrhoeae type IV pilus. To identify cis-acting DNA sequences that facilitate Av, the 700-bp region upstream of the pilin gene pilE was targeted for transposon mutagenesis. Four classes of transposon-associated mutations were isolated, distinguishable by their pilus-associated phenotypes: (i) insertions that did not alter Av or piliation, (ii) insertions that blocked Av, (iii) insertions that interfered with Av, and (iv) insertions that interfered with pilus expression and Av. Mutagenesis of the pilE promoter did not affect the frequency of Av, directly demonstrating that pilin Av is independent of pilE transcription. Two stretches of sequence upstream of pilE were devoid of transposon insertions, and some deletions in these regions were not recoverable, suggesting that they are essential for gonococcal viability. Insertions that blocked pilin Av were located downstream of the RS1 repeat sequence, and deletion of the region surrounding these insertions completely abrogated pilin Av, confirming that specific sequences 5' to pilE are essential for the recombination events underlying pilin Av.

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.