Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.

Staphylococcus aureus infections can be difficult to treat due to both multidrug resistance and the organism's remarkable ability to persist in the host. Persistence and the evolution of resistance may be related to several complex regulatory networks, such as the SOS response, which modifies transcription in response to environmental stress. To understand how S. aureus persists during antibiotic therapy and eventually emerges resistant, we characterized its global transcriptional response to ciprofloxacin. We found that ciprofloxacin induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle. In addition, we found that ciprofloxacin induces the SOS response, which we show, by comparison of a wild-type strain and a non-SOS-inducible lexA mutant strain, includes the derepression of 16 genes. While the SOS response of S. aureus is much more limited than those of Escherichia coli and Bacillus subtilis, it is similar to that of Pseudomonas aeruginosa and includes RecA, LexA, several hypothetical proteins, and a likely error-prone Y family polymerase whose homologs in other bacteria are required for induced mutation. We also examined induced mutation and found that either the inability to derepress the SOS response or the lack of the LexA-regulated polymerase renders S. aureus unable to evolve antibiotic resistance in vitro in response to UV damage. The data suggest that up-regulation of the tricarboxylic acid cycle and induced mutation facilitate S. aureus persistence and evolution of resistance during antibiotic therapy.

The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis. IntroductionBacteria express selected gene systems in a populationdependent manner by sensing self-produced, membranediffusible signals in a strategy called quorum sensing (QS) . The key elements of QS regulation in many Gram-negative bacteria are homologue proteins of LuxI, a N-acyl-homoserinelactone (AHL) signal synthase, and LuxR, an AHL-dependent response regulator. These two proteins control the expression of bioluminescence in the marine bacterium, Vibrio fischeri (Fuqua et al., 1994;Williams et al., 2000;Fuqua et al., 2001;Miller and Bassler, 2001;Withers et al., 2001). Alternative QS mechanisms, mediated by unrelated control factors, exist in other Gram-negative bacteria, most notably Vibrio harveyi (Bassler et al., 1994), and in several Grampositive organisms (Dunny and Leonard, 1997;Kleerebezem and Quadri, 2001). In general, QS governs the control of diverse phenotypes, each benefiting a bacterium in a specialized habitat (Whiteley et al., 1999;Pierson, 2000;Whitehead et al., 2001).Pantoea stewartii ssp. stewartii (P. stewartii) is the causative agent of Stewart's wilt disease in sweetcorn and leaf blight in maize. Disease symptoms develop when the bacterium produces large amounts of a capsular polysaccharide (CPS), which blocks the corn xylem vessels and induces necrotic lesions (Coplin et al., 1992). CPS synthesis is a QS-controlled phenotype governed by the LuxI and LuxR homologue proteins, EsaI and EsaR (von Bodman and Farrand, 1995). Disruption of the signal synthase gene, esaI, leads to parallel loss of AHL, CPS production, and virulence. In contrast, mutations in the esaR gene give a hypermucoid phenotype irrespective of AHL (von Bodman et al., 1998). The simplest explanation for these observations is that EsaR functions as a repressor of CPS synthesis and that derepression requires inducing levels of AHL. The functions required for CPS synthesis are encoded by an extensive cps gene system (Dolph et al., 1988). This gene system is closely related to the wza gene cluster encoding the synthesis of the group I capsules, colanic acid in Escherichia coli (Reeves et al., 1996), and amylovoran in Erwinia

The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo͞capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI͞EsaR quorumsensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI͞EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.maize ͉ xylem ͉ pathogenesis ͉ dissemination

A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. The extent and efficiency of SARS-CoV-2 pandemic is far greater than previous coronaviruses that emerged in the 21st Century. Here, we modeled stability of SARS-CoV-2 on skin, paper currency, and clothing to determine if these surfaces may factor in the fomite transmission dynamics of SARS-CoV-2. Skin, currency, and clothing samples were exposed to SARS-CoV-2 under laboratory conditions and incubated at three different temperatures (4°C± 2°C, 22°C± 2°C, and 37°C ± 2°C). We evaluated stability at 0 hours (h), 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure. SARS-CoV-2 was stable on skin through the duration of the experiment at 4°C (14 days). Virus remained stable on skin for at least 96 h at 22°C and for at least 8h at 37°C. There were minimal differences between the tested currency samples. The virus remained stable on the $1 U.S.A. Bank Note for at least 96 h at 4°C while we did not detect viable virus on the $20 U.S.A. Bank Note samples beyond 72 h. The virus remained stable on both Bank Notes for at least 8 h at 22°C and 4 h at 37°C. Clothing samples were similar in stability to the currency. Viable virus remained for at least 96 h at 4°C and at least 4 h at 22°C. We did not detect viable virus on clothing samples at 37°C after initial exposure. This study confirms the inverse relationship between virus stability and temperature. Furthermore, virus stability on skin demonstrates the need for continued hand hygiene practices to minimize fomite transmission both in the general population as well as in workplaces where close contact is common.

SummaryThe LuxR-type quorum-sensing transcription factor EsaR functions as a repressor of exopolysaccharide (EPS) synthesis in the phytopathogenic bacterium Pantoea stewartii ssp. stewartii . The cell densitydependent expression of EPS is critical for Stewart's wilt disease development. Strains deficient in the synthesis of a diffusible acyl-homoserine lactone inducer remain repressed for EPS synthesis and are consequently avirulent. In contrast, disruption of the esaR gene leads to hypermucoidy and attenuated disease development. Ligand-free EsaR functions as a negative autoregulator of the esaR gene and responds to exogenous acyl-homoserine lactone for derepression. The focus of this study was to define the mechanism by which EsaR governs the expression of the cps locus, which encodes functions required for stewartan EPS synthesis and membrane translocation. Genetic and biochemical studies show that EsaR directly represses the transcription of the rcsA gene. RcsA encodes an essential coactivator for RcsA/ RcsB-mediated transcriptional activation of cps genes. In vitro assays identify an EsaR DNA binding site within the rcsA promoter that is reasonably well conserved with the previously described esaR box. We also describe that RcsA positively controls its own expression. Interestingly, promoter proximal genes within the cps cluster are significantly more acyl-homoserine lactone responsive than genes located towards the middle or 3 ¢ ¢ ¢ ¢ end of the gene cluster. We will discuss a possible role of EsaR-mediated quorum sensing in the differential expression of the cps operon.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

The effects of EBOV evolution on diagnostic assays and therapeutic drugs appear to be low.