For this reason, BIBW2992 cost as predicted by the model, there is little antibiotic variation (73–77 mg l-1 of cephamycin C) at the highest lysine concentration (7.4 g l-1) within the entire cadaverine concentration range under investigation. This is due to the fact that the linear effect of lysine is about thrice stronger than that of this diamine. With respect to lysine combined with putrescine, adding 0.20 g l-1 of this diamine to media containing 3.7 g l-1 of amino acid increased production by approximately 40% as compared to that obtained with medium containing just lysine at the same concentration

(Table 2). On the other hand, adding this diamine to media with higher lysine concentrations (7.4 g l-1) adversely affected production due to the negative effect

stemming from the interaction between the compounds (Figure 4D). Thus, the highest production Selleck BMS202 value predicted for 7.7 g l-1 of lysine combined with 0.13 g l-1 of putrescine is just 76 mg l-1. Similar volumetric production values were obtained with basal culture media containing 7.4 g l-1 of lysine as additive (Figure 2). Martín et al. [43] observed that supplementation with putrescine provided much lower mRNA levels than those obtained with 1,Cell Cycle inhibitor 3-diaminopropane in P. chrysogenum cultures. Despite structural similarity between 1,3-diaminopropane and putrescine, these authors suggest that the positive effect obtained with diamines is probably attributable to the three-carbon structure of diamines. On the other hand, Leitão et al. [32] observed an approximately threefold increase when 0.2 g l-1 of putrescine was added to N. lactamdurans cultures. Figures 5 and 6 show the results of two cultivations in bioreactor using 7.0 g l-1 of lysine combined with 5.2 g l-1 of 1,3-diaminopropane

and 5.3 g l-1of lysine combined with 0.64 g l-1 of alpha-aminoadipic acid. These concentrations, predicted by the models as optimal production conditions, resulted in 190 mg l-1 and 160 mg l-1 of cephamycin C for lysine combined with 1,3-diaminopropane and lysine combined with alpha-aminoadipic acid, respectively. Figure 5 Batch cultivation in agitated and aerated bench-bioreactor for lysine combined with 1,3-diaminopropane. Cephamycin C concentration Lck (CephC), specific production, and biomass; basal medium containing cephamycin C production-enhancing compounds at their optimal values (in parentheses), lysine (7.0 g l-1) and 1,3-diaminopropane (5.2 g l-1) (open symbols); control condition: basal medium without additives (solid symbols). Figure 6 Batch cultivation in agitated and aerated bench-bioreactor for lysine combined with alpha-aminoadipic acid. Cephamycin C concentration (CephC), specific production, and biomass; basal medium containing cephamycin C production-enhancing compounds at their optimal values (in parentheses), lysine (5.3 g.l-1) and alpha-aminoadipic acid (0.6 g.

Results are the average of the motility zones of sixteen Petri dishes per strain. Data was statistically analyzed using one-way ANOVA (p < 0.05). Acknowledgements We thank Rodrigo Vena for assistance with the confocal microscopy Thiazovivin datasheet facility, Microquin for the culture media, Catalina Anderson (INTA Concordia, Argentina), Gastón Alanis and Rubén Díaz Vélez (Proyecto El Alambrado) for the citrus plants, Sebastián Graziati and Diego Aguirre for plant technical

assistance and the Proteomics laboratory from the Biosciences core laboratory, King Abdullah University of Science and Technology, for providing the facility and equipment for gel electrophoresis and mass spectrometry analyses. This work was supported by grants from the Argentine Federal Government: ANPCyT (PICT2010-1507 to NG and PICT2010-0300 to JO) and CONICET (PIP2010-2012 to JO and NG), the Fundación Josefina Prats to CGG and FAF. JO and NG are staff members and TZ, GGS, CGG and FAF are fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). Electronic supplementary material Additional file 1: Figure

S1: Characterization of the hrpB − complemented strain on HR and pathogenicity. (A) Schematic organization of the hrp cluster of X. citri that was constructed based on the X. citri subsp. citri strain 306 genome sequence [1]. Boxes correspond to ORFs, arrows RG7112 indicate orientation of the hrp operons. The hrp, hpa and hrc genes are indicated. Dotted boxes indicated the genomic regions replaced by mutagenesis. Bellow of the scheme, the black box represents the genomic fragment cloned in pBBR1MCS-5 to complement the hrpB − mutant strain. (B) Bacterial suspensions of X. citri,

the hrpB − mutant and the hrpB − c strains were inoculated at 108 CFU/ml into the intercellular spaces of fully expanded tomato, cotton and pepper leaves. A representative photograph of a leaf is shown after 1 day of inoculation. (C) As in B, bacterial suspensions at 107 CFU/ml were inoculated into the intercellular spaces of fully expanded citrus leaves. A representative photograph of a leaf is shown after 8 days of inoculation. (D) RT-qPCR to determine Fossariinae CsLOB1 expression levels in leaves after 48 hours of infection with X. citri, the hrpB − mutant and hrpB − c strain. Bars indicate the expression levels relative to buffer infiltrations. Values are the means of four biological replicates with three technical replicates each. (PDF 137 KB) Additional file 2: NVP-BSK805 Figure S2: Swimming and swarming assays. Representative photographs of Petri dishes with X. citri, the hrp mutants and the hrpB − c strain after 2 days of inoculation. Scale bars: 10 mm. (PDF 819 KB) Additional file 3: Table S1: Oligonucleotides used in RT-qPCR assays. (PDF 7 KB) References 1.