Laura Marri

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2)

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. σH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of σH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2–egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild‐type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and σH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2–egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter‐binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of σH is subject to temporal and spatial regulation during colony development, that this tissue‐specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Bacteria associated with the oesophageal bulb of the medfly Ceratitis capitata (Diptera:Tephritidae)

Extracellular Gram negative bacteria were found to be commonly associated to the oesophageal bulb of Ceratitis capitata with Klebsiella oxytoca and Enterobacter agglomerans as the most common species. All the isolates tested in vitro, except one, were sensitive to the antibacterial material present on the medfly laid egg surface.

The Novel Antibacterial Peptide Ceratotoxin A Alters Permeability of the Inner and Outer Membrane of Escherichia coli K-12

Abstract. Ceratotoxins are antibacterial 3-kDa amphiphilic peptides isolated from the female reproductive apparatus of the medfly Ceratitis capitata. The antibacterial activity of a chemically synthesized ceratotoxin A (ctx A) has been investigated. Ctx A was mainly active against Gram-negative organisms, and it had a lytic effect on nongrowing Escherichia coli K-12. Data showed that ctx A alters both the outer and the inner membrane of E. coli K-12 cells.

Alicyclobacillus pohliae sp. nov., a thermophilic, endospore-forming bacterium isolated from geothermal soil of the north-west slope of Mount Melbourne (Antarctica).

Gram-positive, rod-shaped, endospore-forming, thermophilic bacteria were isolated from a geothermal soil collected on the north-west slope of Mount Melbourne in Antarctica. They grew aerobically at 42-60 degrees C (optimum 55 degrees C) and at pH 4.5-7.5 (optimum pH 5.5). Phylogenetic analysis of 16S rRNA gene sequences showed that these isolates were related most closely to the type strain of Alicyclobacillus pomorum (91% similarity). Growth occurred in the presence of ferrous iron at micromolar concentrations and acid was produced from various sugars. Iso-branched fatty acids C(15:0) (45.56%) and C(17:0) (35.81%) were the most abundant cellular fatty acids. The DNA G+C content was 55.1 mol%. On the basis of phenotypic and phylogenetic characteristics, it is concluded that these strains represent a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus pohliae sp. nov. is proposed. The type strain is MP4(T) (=CIP 109385(T) =NCIMB 14276(T)).

The female reproductive accessory glands of the medfly Ceratitis capitata : antibacterial activity of the secretion fluid

Abstract Secretion from female reproductive accessory glands of the dipteran Ceratitis capitata was found to have antibacterial properties against E. coli . At least two basic polypeptides with mol. wt 15.5 and 4.7 kDa respectively, were identified as responsible for such activity. Furthermore, the 15.5 kDa protein is active against a number of Gram-positive and -negative bacterial strains. Lysozyme activity is also present in the secretion.

Pathogenic Mechanisms of Helicobacter pylori: Production of Cytotoxin

Bacteria associated with mucosal infections of the digestive system generally produce toxins, especially when they cause inflammatory lesions. Illnesses due to thermotolerant campylobacters, to enterohemorrhagic Escherichia coli, and to Clostridium difficileare only some examples. It would be surprising if Helicobacter pylori (HP) did not produce any toxic substances. The difficulty consists in attributing a pathogenic meaning to the toxin, since the range is quite wide of clinical and histological presentation of gastroduodenal inflammatory diseases linked to the presence in the stomach of H. pylori organisms [1]. Johnson and Lior [2] firstly reported the production of heat-labile cytotoxin by 80.6% of 36 HP strains they tested. However, most of our knowledge of the cytotoxigenicity of HP is from Leunk et al. [3] whose work has inspired us in part. They found that about 55% of 201 HP strains isolated in four different parts of the world produced a substance which caused intracellular vacuolization in cells of several lines in vitro, not only in lines generally employed in toxigenicity tests, like Chinese hamster ovary (CHO) cells, Vero cells, and Y-1 cells, but also in human tumoral cells like HeLa, KATO III, and HEp-2, as well as in human embryonic intestinal cells which were the most responsive. They also inferred that the toxin was proteinaceous in nature being heat labile (destroyed at 70 °C for 30 min), protease sensitive, and ammonium-sulfate precipitable. Its molecular weight ought to be higher than 100 kDa since cytotoxic activity could be found only in the retentate of a concentrated broth culture filtrate (CBCF) passed through 100 kDa molecular weight limit ultrafiltration membrane.

Iridovirus infection in terrestrial isopods from Sicily (Italy)

During our researches on systematics and ecology of terrestrial isopods, carried out in western Sicily, some specimens showing a blue-purple coloration were collected; they belonged to four species: Armadillidium decorum Brandt, 1833, Trichoniscus panormidensis Montesanto et al., 2011, Philoscia affinis Verhoeff, 1908, Porcellio siculoccidentalis Viglianisi et al., 1992. We hypothesized that such coloration could be due, as reported in literature, to characteristic paracrystalline arrays of virions inside the tissues of blue colored specimens. Ultrastructural observations by transmission electron microscopy, on tissues of A. decorum, showed the presence of electron-dense viral particles, with a diameter of nearly 0.12μm. Dual-axis tomography, performed on specimens of A. decorum, evidenced an icosahedral structure of viral particles matching with that of Isopod Iridescent Virus (IIV). Molecular analysis, on 254bp portion of the major capsid protein (MCP) gene, allowed to place the virus into IIV-31 group, already known for other oniscidean species. The symptoms of infected individuals and the course of the disease were followed in laboratory, indicating similarities with other studies on Isopod Iridoviruses. Moreover, some notes on reproduction of infected ovigerous females are reported. Our data support unequivocal and direct evidences for the first case of IIV infection in terrestrial isopods reported in Italy.

The antibacterial peptide ceratotoxin A displays alamethicin-like behavior in lipid bilayers.

Ceratotoxin A (CtxA), a 36-residue alpha-helical cationic peptide isolated from the medfly Ceratitis capitata, exhibits strong antibacterial activity. To determine its mode of action against bacteria, we investigated the behavior of ceratotoxin A by incorporating it into planar lipid bilayers. Macroscopic and single channel conductance experiments showed that ceratotoxin A forms voltage-dependent ion channels in bilayers according to the barrel-stave model. The characteristics of the channel suggest that the C-terminal regions form bundles of five or six helices embedded in the membrane, such that the N-terminal moieties lie on the polar side of the lipid bilayer.

Transformation as a tool for studying the epidemiology of tet determinants in Streptococcus pneumoniae.

Transformation of pneumococcus was used to detect homology among tetracycline resistance determinants of clinical isolates of Streptococcus pneumoniae. A strain of pneumococcus containing a mutated tet determinant (tet-3), of class M, integrated into the chromosome was used as a recipient in transformation experiments, where donor DNA was from the tetracycline resistant isolates. 34/34 strains appeared to have tet determinants homologous to tet-3 (i.e. tet M).Still using transformation it was possible to determine that the tet-3 transforming activity of DNA from Tn916 and S. pneumoniae BM6001 was contained in a 5 kb HincII fragment. For this purpose a transformation technique where donor DNA was directly taken from low melting point agarose gels was standardized and used.

Green fluorescent protein (GFP)-labeling of enterobacteria associated with fruit flies (Diptera: Tephritidae) and persistence in their natural host Rhagoletis completa Cresson.

Fruit flies (Diptera: Tephritidae) are a highly successful, widespread group of insects that cause economic damage in agriculture. Data available so far on the composition of the bacterial community associated with their digestive tract indicate that members of Enterobacteriaceae are the species most often isolated. Bacteria naturally occurring in insect guts may be engineered and used to study the spatial and functional interactions of microbes within the insect system and offer one route to meet the demand for novel insect pest management strategies. With this aim we introduced by conjugation the gfp gene carried by the suicide plasmid pTn5gfpmut1 into Klebsiella oxytoca and Raoultella (formerly Klebsiella ) spp. strains isolated from the oesophageal bulb of the fruit flies Ceratitis capitata (Wiedemann) and Rhagoletis completa Cresson, respectively. The GFP-encoding gene was stably maintained in two tested transgenic strains, both originally isolated from R. completa. In one case, GFP-labeled bacterial cells were used to feed larvae and adults of the original host. Genetically modified bacteria were able to colonize the gut of larvae and persisted through all larval instars to pupal stage.