Teresa M. Barbosa

Differential Expression of over 60 Chromosomal Genes in Escherichia coli by Constitutive Expression of MarA

In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.

Characterization of Bacillus Probiotics Available for Human Use

ABSTRACT Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

Screening for bacillus isolates in the broiler gastrointestinal tract.

ABSTRACT Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.

Occurrence of the New Tetracycline Resistance Gene tet(W) in Bacteria from the Human Gut

ABSTRACT Members of our group recently identified a new tetracycline resistance gene, tet(W), in three genera of rumen obligate anaerobes. Here, we show that tet(W) is also present in bacteria isolated from human feces. The tet(W) genes found in human Fusobacterium prausnitzii andBifidobacterium longum isolates were more than 99.9% identical to those from a rumen isolate of Butyrivibrio fibrisolvens.

Diversity and antimicrobial activity of Pseudovibrio spp. from Irish marine sponges.

Aims:  To evaluate the diversity and antimicrobial activity present among Pseudovibrio spp. isolated from marine sponges.

Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in a class II promoter

MarA is a global regulator that mediates resistance to multiple environmental hazards such as antibiotics, disinfectants and oxidative stress agents by modulating the expression of a large number of genes in the Escherichia coli chromosome. Two E. coli MarA homologues, SoxS and Rob also control, to different extents, genes in the mar/sox/rob regulon. The controlling element for these proteins is a 20 bp ‘marbox’ sequence in the promoter region of regulated genes. Using in vitro assays and mutagenesis of promoter fusions in whole cells, we identified the cis regulatory element involved in MarA upregulation of the oxygen‐insensitive nitroreductase nfnB gene. MarA binds to a marbox that is highly divergent from the previously proposed consensus (eight differences out of 14 specified nucleotides). Although purified SoxS and Rob proteins, like MarA, activated nfnB transcription in vitro, only constitutive expression of chromosomal marA, but not of soxS and rob genes, affected nfnB expression in whole cells. Increased expression, but limited as compared with MarA, was only achieved by plasmid‐mediated overexpression of SoxS and Rob. This study shows that MarA can regulate gene expression through a functional marbox that is considerably divergent from the current consensus sequence. The data suggest that MarA is preferred over SoxS and Rob in upregulating nfnB. The findings imply that other different but physiologically important marbox DNA–MarA interactions take place in the regulation of still uncharacterized members of the mar regulon.

The Escherichia coli Transcriptional Regulator MarA Directly Represses Transcription of purA and hdeA

The Escherichia coli MarA protein mediates a response to multiple environmental stresses through the activation or repression in vivo of a large number of chromosomal genes. Transcriptional activation for a number of these genes has been shown to occur via direct interaction of MarA with a 20-bp degenerate asymmetric “marbox” sequence. It was not known whether repression by MarA was also direct. We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA. Transcription and electrophoretic mobility shift experiments in vitro using mutant promoters suggested that the marbox involved in the repression overlapped the -35 promoter motif and was in the “backward” orientation. This organization contrasts with that of the class II promoters activated by MarA, in which the marbox also overlaps the -35 motif but is in the “forward” orientation. We conclude that MarA, a member of the AraC/XylS family, can act directly as a repressor or an activator, depending on the position and orientation of the marbox within a promoter.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans

Aims:  Despite the frequent isolation of endospore‐formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore‐forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds.

Tetracycline Resistance-Encoding Plasmid from Bacillus sp. Strain #24, Isolated from the Marine Sponge Haliclona simulans

ABSTRACT Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.