The Phaseolus vulgaris Receptor-Like Kinase PvFER1 and the Small Peptides PvRALF1 and PvRALF6 Regulate Nodule Number as a Function of Nitrate Availability.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Actin Depolymerizing Factor Modulates Rhizobial Infection and Nodule Organogenesis in Common Bean.

Actin plays a critical role in the rhizobium-legume symbiosis. Cytoskeletal rearrangements and changes in actin occur in response to Nod factors secreted by rhizobia during symbiotic interactions with legumes. These cytoskeletal rearrangements are mediated by diverse actin-binding proteins, such as actin depolymerization factors (ADFs). We examined the function of an ADF in the Phaseolus vulgaris-rhizobia symbiotic interaction (PvADFE). PvADFE was preferentially expressed in rhizobia-inoculated roots and nodules. PvADFE promoter activity was associated with root hairs harbouring growing infection threads, cortical cell divisions beneath root hairs, and vascular bundles in mature nodules. Silencing of PvADFE using RNA interference increased the number of infection threads in the transgenic roots, resulting in increased nodule number, nitrogen fixation activity, and average nodule diameter. Conversely, overexpression of PvADFE reduced the nodule number, nitrogen fixation activity, average nodule diameter, as well as NODULE INCEPTION (NIN) and EARLY NODULIN2 (ENOD2) transcript accumulation. Hence, changes in ADFE transcript levels affect rhizobial infection and nodulation, suggesting that ADFE is fine-tuning these processes.

The 95ΔG mutation in the 5'untranslated region of the norA gene increases efflux activity in Staphylococcus epidermidis isolates.

In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 µg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR 95ΔG mutation. Isolates carrying the 95ΔG mutation had higher levels of norA expression compared with those that did not. To corroborate that the 95ΔG mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the 95ΔG mutation and had a high level of norA expression and EF, indicating that the 95ΔG mutation is important for the HEA phenotype. The 95ΔG mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR 95ΔG mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.

Identification and expression of nor efflux family genes in Staphylococcus epidermidis that act against gatifloxacin.

NorA, NorB, and NorC are efflux proteins in the Nor family that regulate the secretion of fluoroquinolones, and MgrA/NorR is a transcription factor of the Nor family. Overexpression of Nor family proteins provides fluoroquinolone resistance in Staphylococcus aureus. However, in coagulase-negative staphylococci (CNS), members of the Nor family had not been identified. In this work, the presence of Nor family proteins in Staphylococcus spp. and the expression of Nor family in gatifloxacin resistant S. epidermidis strains obtained from ocular infections (OI) were identified and analyzed. S. epidermidis strains from OIs (n = 44) and healthy skin (HS; n = 52) were isolated. The nor family genes were identified in CNS using PCR, sequencing and phylogenetic approaches. Nor family expression was determined by RT-PCR. NorA efflux activity was determined using the automated ethidium bromide method. In-silico analysis showed that norA, mgrA/norR, and "norB-like" and "norC-like" (norB/norC) genes are present in CNS. The nor family genes were distributed and constitutively expressed in all S. epidermidis strains studied. In one gatifloxacin resistant strain isolated from the endophthalmitis, treatment with gatifloxacin induced overexpression of the norA gene and resulted in high activity of NorA efflux. These results indicate that the Nor family of proteins is present in CNS, and the NorA efflux mechanism for gatifloxacin response occurs in at least one strain of S. epidermidis, contributing to gatifloxacin resistance.

Detection of hssS, hssR, hrtA, and hrtB genes and their expression by hemin in Staphylococcus epidermidis.

Staphylococcus aureus employs a heme sensing system (HssR-HssS) and a heme-regulated transporter efflux pump (HrtA-HrtB) to avoid the accumulation of heme, which is toxic at high concentrations. The detoxification system to heme has not been studied in Staphylococcus epidermidis . In this work, the hssR, hssS, hrtA, and hrtB genes were detected, and their expression when stimulated by hemin in S. epidermidis was explored. In silico genomic analyses exhibited that the genetic organization of the hssRS and hrtAB genes was identical in 11 Staphylococcus species analyzed, including S. epidermidis. Slight variations were found in their syntenic regions. The predicted secondary structure of HrtAB proteins from these species was almost identical to these of S. aureus. Additionally, hrtAB promoter sequences of some species were analyzed, and 1 or 2 different nucleotide substitutions were found in the downstream motif. Concentrations of hemin above 5 µmol/L inhibited S. epidermidis growth. However, S. epidermidis that was pre-exposed to a subinhibitory hemin concentration (1 µmol/L) was able to grow when inoculated into medium containing above 5 µmol/L hemin. The expression levels of hrtA and hrtB genes in S. epidermidis exhibited a significant difference when they were stimulated with hemin. Our results suggest that the HrtAB could be involved in hemin detoxification of S. epidermidis.

Gatifloxacin, moxifloxacin, and balofloxacin resistance due to mutations in the gyrA and parC genes of Staphylococcus epidermidis strains isolated from patients with endophthalmitis, corneal ulcers and conjunctivitis.

AIMS: Staphylococcus epidermidis is considered a commensal bacterium; however, it is frequently isolated from ocular infections showing a multidrug resistance. Ciprofloxacin-resistant strains have been isolated from ocular infections; however, resistance to quinolone, such as gatifloxacin and moxifloxacin, is not often studied, consequently the resistance mechanism is unknown. Our aim was to address the quinolone resistance and to explore the resistance mechanism in S. epidermidis strains isolated from ocular infections. METHODS: S. epidermidis strains were isolated from patients with conjunctivitis (n = 23), endophthalmitis (n = 14) and corneal ulcers (n = 7). Minimum inhibition concentrations were determined by broth and agar dilution methods for moxifloxacin, gatifloxacin, balofloxacin, rufloxacin and pazufloxacin. Mutations were identified by sequencing the gyrA and parC genes, and their expression was determined by reverse transcriptase polymerase chain reaction. RESULTS: We found that 13.6% (6/44) of the strains were quinolone resistant. In endophthalmitis, 21.4% were gatifloxacin, moxifloxacin and balofloxacin resistant. In corneal ulcers, 14.2, 14.2 and 28.5% were gatifloxacin, moxifloxacin and balofloxacin resistant, respectively, and in conjunctivitis only 4.3% were gatifloxacin resistant. The 6 strains with quinolone resistance showed mutations at Ser84Phe for the gyrA gene, and Ser80Phe for the parC gene. Gatifloxacin did not change the expression levels of gyrA and parC genes. CONCLUSION: S. epidermidis strains isolated from three ocular pathologies were gatifloxacin and moxifloxacin resistant due to mutations on the gyrA and parC genes.

The NALP3/Cryopyrin-inflammasome complex is expressed in LPS-induced ocular inflammation.

In the inflammosome complex, NALP3 or NALP1 binds to ASC and activates caspase-1 which induces IL-1beta. In murine LPS-induced ocular inflammation, the production of IL-1beta is increased. We suggest that NALP3- or NALP1-inflammasome complex can be participating in the LPS-induced ocular inflammation. In this work, eye, brain, testis, heart, spleen, and lung were obtained from C3H/HeN mice treated with LPS for 3 to 48 hours, and the expression of NALP1b, NALP3, ASC, caspase-1, IL-1beta, and IL-18 was determined. Infiltrated leukocytes producing IL-1beta in the anterior chamber were found at 12-hour posttreatment. A high upregulated expression of NALP3, ASC, caspase-1, IL-1beta, and IL-18 was found at the same time when infiltrated leukocytes were observed. NALP1b was not detected in the eye of treated mice. NALP3 was also overexpressed in heart and lung. These results suggest that NALP3-, but not NALP1-inflammosome complex, is participating in the murine LPS-induced ocular inflammation.

The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules.

Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

Staphylococcus epidermidis with the icaA⁻/icaD⁻/IS256⁻ genotype and protein or protein/extracellular-DNA biofilm is frequent in ocular infections.

In ocular infections (OIs) caused by Staphylococcus epidermidis, biofilms composed mainly of poly-N-acetylglucosamine (PNAG) have been widely studied, but PNAG-independent biofilms have not. Therefore, we searched for a relationship between the ica operon (involved in PNAG-biofilm) and the biochemical composition of biofilms in isolates from OI. Isolates from OI (n = 62), from healthy conjunctiva (HC; n = 45) and from healthy skin (HS; n = 53), were used to detect icaA and icaD genes, and the insertion sequence 256 (IS256) using PCR. The compositions of the biofilms were determined by treatment with NaIO4, proteinase K and DNase I. Multilocus sequence typing (MLST) was performed to characterize the isolates, and the expression of aap and embp genes was determined by real-time qPCR. A strong relationship between the icaA(-)/icaD(-)/IS256(-) genotype and protein- or protein/extracellular DNA (eDNA)-biofilm composition was found in the isolates from OI (53.6%), whereas the icaA(+)/icaD(+)/IS256(-) genotype and carbohydrate-biofilm was most prevalent in isolates from HC (25%) and HS (25%). Isolates with an icaA(-)/icaD(-)/IS256(-) genotype and protein-biofilm phenotype were predominantly of the ST2 lineage, while carbohydrate-biofilm-producing strains were mainly of the ST9 lineage. The protein-biofilm-producing strains had higher expression levels of aap gene than carbohydrate-biofilm-producing strains; while embp gene did not have the same pattern of expression. These results suggest that S. epidermidis strains with icaA(-)/icaD(-)/IS256(-) genotype and protein- or protein/eDNA-biofilms have a stronger ability to establish in the eye than S. epidermidis strains with icaA(+)/icaD(+)/IS256(-) genotype and PNAG-biofilms.