Summary: Extracellular polysaccharides comprise a major component of the biofilm matrix. Many species that are adept at biofilm formation have the capacity to produce multiple types of polysaccharides. Pseudomonas aeruginosa produces at least three extracellular polysaccharides, alginate, Pel and Psl, that have been implicated in biofilm development. Non‐mucoid strains can use either Pel or Psl as the primary matrix structural polysaccharide. In this study, we evaluated a range of clinical and environmental P. aeruginosa isolates for their dependence on Pel and Psl for biofilm development. Mutational analysis demonstrates that Psl plays an important role in surface attachment for most isolates. However, there was significant strain‐to‐strain variability in the contribution of Pel and Psl to mature biofilm structure. This analysis led us to propose four classes of strains based upon their Pel and Psl functional and expression profiles. Our data also suggest that Pel and Psl can serve redundant functions as structural scaffolds in mature biofilms. We propose that redundancy could help preserve the capacity to produce a biofilm when exopolysaccharide genes are subjected to mutation. To test this, we used PAO1, a common lab strain that primarily utilizes Psl in the matrix. As expected, a psl mutant strain initially produced a poor biofilm. After extended cultivation, we demonstrate that this strain acquired mutations that upregulated expression of the Pel polysaccharide, demonstrating the utility of having a redundant scaffold exopolysaccharide. Collectively, our studies revealed both unique and redundant roles for two distinct biofilm exopolysaccharides.

Summary: Nitric oxide (NO·) is an important mediator of innate immunity. The facultative intracellular pathogen Salmonella has evolved mechanisms to detoxify and evade the antimicrobial actions of host‐derived NO· produced during infection. Expression of the NO·‐detoxifying flavohaemoglobin Hmp is controlled by the NO·‐sensing transcriptional repressor NsrR and is required for Salmonella virulence. In this study we show that NsrR responds to very low NO· concentrations, suggesting that it plays a primary role in the nitrosative stress response. Additionally, we have defined the NsrR regulon in Salmonella enterica sv. Typhimurium 14028s using transcriptional microarray, qRT‐PCR and in silico methods. A novel NsrR‐regulated gene designated STM1808 has been identified, along with hmp, hcp‐hcr, yeaR‐yoaG, ygbA and ytfE. STM1808 and ygbA are important for S. Typhimurium growth during nitrosative stress, and the hcp‐hcr locus plays a supportive role in NO· detoxification. ICP‐MS analysis of purified STM1808 suggests that it is a zinc metalloprotein, with histidine residues H32 and H82 required for NO· resistance and zinc binding. Moreover, STM1808 and ytfE promote Salmonella growth during systemic infection of mice. Collectively, these findings demonstrate that NsrR‐regulated genes in addition to hmp are important for NO· detoxification, nitrosative stress resistance and Salmonella virulence.

Bioaerosols are emerging as important yet poorly understood players in atmospheric processes. Microorganisms can impact atmospheric chemistry through metabolic reactions and can potentially influence physical processes by participating in ice nucleation and cloud droplet formation. Microbial roles in atmospheric processes are thought to be species-specific and potentially dependent on cell viability. Using a coastal pier monitoring site as a sampling platform, culture-dependent (i.e. agar plates) and culture-independent (i.e. DNA clone libraries from filters) approaches were combined with 18S rRNA and 16S rRNA gene targeting to obtain insight into the local atmospheric microbial composition. From 13 microbial isolates and 42 DNA library clones, a total of 55 sequences were obtained representing four independent sampling events. Sequence analysis revealed that in these coastal samples two fungal phyla, Ascomycota and Basidiomycota, predominate among eukaryotes while Firmicutes and Proteobacteria predominate among bacteria. Furthermore, our culture-dependent study verifies the viability of microbes from all four phyla detected through our culture-independent study. Contrary to our expectations and despite oceanic air mass sources, common marine planktonic bacteria and phytoplankton were not typically found. The abundance of terrestrial and marine sediment-associated microorganisms suggests a potential importance for bioaerosols derived from beaches and/or coastal erosion processes.

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5′ GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3′ end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5′ splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

The M1T1 clone of group A Streptococcus (GAS) is associated with severe invasive infections, including necrotizing fasciitis and septicemia. During invasive M1T1 GAS disease, mutations in the covRS regulatory system led to upregulation of an ADP-ribosyltransferase, SpyA. Surprisingly, a GAS Δ spyA mutant was resistant to killing by macrophages and caused higher mortality with impaired bacterial clearance in a mouse intravenous challenge model. GAS expression of SpyA triggered macrophage cell death in association with caspase-1-dependent interleukin 1β (IL-1β) production, and differences between wild-type (WT) and Δ spyA GAS macrophage survival levels were lost in cells lacking caspase-1, NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), or pro-IL-1β. Similar in vitro findings were identified in macrophage studies performed with pseudomonal exotoxin A, another ADP-ribosylating toxin. Thus, SpyA triggers caspase-1-dependent inflammatory cell death in macrophages, revealing a toxin-triggered IL-1β-dependent innate immune response pathway critical in defense against invasive bacterial infection. IMPORTANCE Group A Streptococcus (GAS) is a leading human pathogen capable of producing invasive infections even in healthy individuals. GAS bacteria produce a toxin called SpyA that modifies host proteins through a process called ADP ribosylation. We describe how macrophages, frontline defenders of the host innate immune system, respond to SpyA by undergoing a specialized form of cell death in which they are activated to release the proinflammatory cytokine molecule interleukin 1β (IL-1β). Release of IL-1β activates host immune cell clearance of GAS, as we demonstrated in tissue culture models of macrophage bacterial killing and in vivo mouse infectious-challenge experiments. Similar macrophage responses to a related toxin of Pseudomonas bacteria were also shown. Thus, macrophages recognize certain bacterial toxins to activate a protective immune response in the host.