Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosis dramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.

Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis . Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS 6110 . Here, we mapped and sequenced the regions flanking the two copies of IS 6110 in the B strain. Ligation-mediated PCR showed that one of these IS 6110 copies is located within the promoter region of phoP , a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS 6110 in the phoP promoter. To determine whether IS 6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS 6110 from the B strain and the equivalent region from M. tuberculosis without IS 6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS 6110 , as in the case of the B strain.

The attenuatedMycobacterium tuberculosisH37Ra strain is an isogenic counterpart of the virulent paradigm strain H37Rv. Recently, a link between a point mutation in the PhoP transcriptional regulator and avirulence of H37Ra was established. Remarkably, a previous study demonstrated negative autoregulation of thephoPgene in H37Ra. These findings led us to study the transcriptional autoregulation of PhoP in the virulent H37Rv strain. In contrast to the negative autoregulation of PhoP previously published for H37Ra, our experiments using aphoPpromoter-lacZfusion showed that PhoP is positively autoregulated in both H37Rv and H37Ra compared with an H37RvphoPdeletion mutant constructed in this study. Using quantitative reverse transcription-PCR (RT-PCR) analysis, we showed that thephoPgene is transcribed at similar levels in H37Rv and H37Ra. Gel mobility shift and DNase I footprinting assays allowed us to identify the precise binding region of PhoP from H37Rv to thephoPpromoter. We also carried out RT-PCR studies to demonstrate thatphoPis transcribed together with the adjacent genephoR, which codes for the cognate histidine kinase of thephoPRtwo-component system. In addition, quantitative RT-PCR studies showed thatphoRis independently transcribed from a promoter possibly regulated by PhoP. Finally, we discuss the possible role in virulence of a single point mutation found in thephoPgene from the attenuated H37Ra strain but not in virulent members of theM. tuberculosiscomplex.

Berries of Vaccinium meridionale Swartz contain a variety of phytochemicals, which are believed to account for their bioactive properties. The potential of Vaccinium meridionale Swartz pomace as a source of bioactive compounds was investigated. The dietary fiber (DF) content was assessed by the AOAC method, phenolic compounds were characterized and quantified via HPLC-PDA and UPLC-QTOF-MS. The in vitro antibacterial activity was tested against Gram-positive and Gram-negative bacteria. The antioxidant properties were assessed by the ORAC and the ABTS assays. The DF content was 52.4 ± 3.7%, phenolic compounds comprised anthocyanins (ACNs) (747.6 ± 167.5 mg cyanidin-3-glucoside/100 g FW), hydroxycinammic acids (HCAs) (229.2 ± 68.4 mg chlorogenic acid equivalents/100 g FW), flavonols (335.0 ± 139.5 rutin equivalents/100 g FW), and procyanidins (PACs) (140.9 ± 33.3 mg cocoa procyanidin equivalents/100 g FW). Staphylococcus aureus was more sensitive than E. coli. The ORAC value was 250.0 ± 32.0 μmol TE/g fresh weight (FW). Results suggest that the residue from V. meridionale S. can be utilized to obtain valuable nutraceuticals for the development of functional foods.

Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals' diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE-PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment.