Inactivation of the ybdD gene in Lactococcus lactis increases the amounts of exported proteins.

Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a protein-overproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.

Production of lipopeptides among Bacillus strains showing growth inhibition of phytopathogenic fungi.

The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance to eliminate chemical pollutants.

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp.

AIMS: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. METHODS AND RESULTS: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 °C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. CONCLUSIONS: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. SIGNIFICANCE AND IMPACT OF THE STUDY: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Aminonaphthoquinone induces oxidative stress in Staphylococcus aureus.

The biological activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) on Staphylococcus aureus was investigated in comparison with the unsubstituted 1,4-naphthoquinone (NQ). Complete inhibition of microbial growth was observed with ANQ and NQ at 50 and 10 microg/mL, respectively. The antibacterial effect of naphthoquinones decreased in the presence of sodium ascorbate, but the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was able to protect S. aureus only from the harmful effect of ANQ. Naphthoquinones blocked oxygen uptake and induced cyanide-insensitive oxygen consumption. When combining rotenone or salicylhydroxamic acid with ANQ or NQ, a slight decrease in respiratory activity was observed. Assays in the presence of naphthoquinones induced an increase of lipid peroxidation in S. aureus, as determined by thiobarbituric acid reactive substances. These results showed that 1,4-naphthoquinones effectively act as electron acceptors and induce an increase in reactive oxygen species that are toxic to S. aureus cells.

An unusual reduction on the quinonoid ring of 5-amino-8-hydroxy-1,4-naphthoquinone by Staphylococcus aureus.

AIMS: The objective of this study was to investigate the interactions between 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and Staphylococcus aureus. METHODS AND RESULTS: The compound ANQ display antimicrobial activity against S. aureus. During incubation with 50 microg ml(-1) of ANQ, an unusual reduction reaction takes place and leads to the isolation of 2,3-dihydro-5-amino-8-hydroxy-1,4-naphthoquinone (ANQ-H(2)), fully characterized by means of (13)C-NMR and (1)H-NMR, plus infrared, UV-visible and mass spectroscopy. Oxygen uptake by S. aureus cells was inhibited by ANQ, but in a significantly minor extent by ANQ-H(2). CONCLUSIONS: The ability of S. aureus to reduce the double bond at C2-C3 of the ANQ is a unusual behaviour for biological transformation of naphthoquinones. SIGNIFICANCE AND IMPACT OF THE STUDY: This uncommon reaction may provide valuable understanding of the S. aureus regarding to the antimicrobial effect and the acquisition of resistance to naphthoquinones.