Lucía Feijoo-Siota

Biodegradation of Naphthalene by Pseudomonas stutzeri in Marine Environments: Testing Cells Entrapment in Calcium Alginate for Use in Water Detoxification

ABSTRACT The biodegradation of naphthalene in sea water by freely suspended and alginate-entrapped cells of Pseudomonas stutzeri 19SMN4 has been investigated in batch cultures. The results showed that immobilized cells can be stored at 4°C for 1 month without loss of viability. The biodegradation was highly affected by the availability of nitrogen and phosphorous, so at 30°C a naphthalene concentration of 25 mM was almost completely degraded (93%) by free cells in 6 days in samples supplemented with these nutrients, whereas only 42% naphthalene was consumed in the nonsupplemented samples. Biodegradation was much slower at 16°C than at 30°C; after 6 days of culture at 30°C, almost all naphthalene was degradated by free and immobilized cells, whereas only 22% and 34% at 16°C, respectively. The degradation rate remained unaffected when the naphthalene concentration was reduced from 25 to 10 mM. Alginate of three different viscosities was used for immobilization of cells. After 7 days of culture, beads formed with 31.4 cP alginate were fragmented, whereas beads formed with 240 and 3600 cP did not display structural changes and afforded the same degradation rate. Beads formed with high-viscosity alginate retained cells more efficiently.

Recent patents on microbial proteases for the dairy industry.

This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.

Proposal of a method for the genetic transformation of Gordonia jacobaea

Aims:  Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans‐canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea.

Considerations on bacterial nucleoids

The classic genome organization of the bacterial chromosome is normally envisaged with all its genetic markers linked, thus forming a closed genetic circle of duplex stranded DNA (dsDNA) and several proteins in what it is called as “the bacterial nucleoid.” This structure may be more or less corrugated depending on the physiological state of the bacterium (i.e., resting state or active growth) and is not surrounded by a double membrane as in eukayotic cells. The universality of the closed circle model in bacteria is however slowly changing, as new data emerge in different bacterial groups such as in Planctomycetes and related microorganisms, species of Borrelia, Streptomyces, Agrobacterium, or Phytoplasma. In these and possibly other microorganisms, the existence of complex formations of intracellular membranes or linear chromosomes is typical; all of these situations contributing to weakening the current cellular organization paradigm, i.e., prokaryotic vs eukaryotic cells.

Antivirals against animal viruses

Abstract Antivirals are compounds used since the 1960s that can interfere with viral development. Some of these antivirals can be isolated from a variety of sources, such as animals, plants, bacteria or fungi, while others must be obtained by chemical synthesis, either designed or random. Antivirals display a variety of mechanisms of action, and while some of them enhance the animal immune system, others block a specific enzyme or a particular step in the viral replication cycle. As viruses are mandatory intracellular parasites that use the host’s cellular machinery to survive and multiply, it is essential that antivirals do not harm the host. In addition, viruses are continually developing new antiviral resistant strains, due to their high mutation rate, which makes it mandatory to continually search for, or develop, new antiviral compounds. This review describes natural and synthetic antivirals in chronological order, with an emphasis on natural compounds, even when their mechanisms of action are not completely understood, that could serve as the basis for future development of novel and/or complementary antiviral treatments.

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris

Galium verum, also known as Ladys Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and β-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

A short voyage into the past: former misconceptions and misinterpretations in the etiology of some viral diseases

The advancement of human knowledge has historically followed the pattern of one-step growth (the same pattern followed by microorganisms in laboratory culture conditions). In this way, each new important discovery opened the door to multiple secondary breakthroughs, eventually reaching a “plateau” when new findings emerged. Microbiology research has usually followed this pattern, but often the conclusions attained from experimentation/observation were either equivocal or altogether false, causing important delays in the advancement of this science. This mini-review deals with some of these documented scientific errors, but the aim is not to include every mistake, but to select those that are paramount to the advance of Microbiology.

Fecal Matter Implantation as a Way to Fight Diarrhea-Causing Microorganisms

This chapter summarizes the current knowledge about fecal microbiota transplantation (FMT) as a way to treat persistent diarrhea. Although this is an old concept, originated in China at least a millennium ago, it also represents a novel approach and an alternative to the use of antibiotics. This approach is a major departure from Paul Ehrlich´s old paradigm ‘the magic bullet,’ or a compound that supplied in a ‘dossa sterilisa magna’ would eliminate the parasite protozoan or bacterium, and represents a more ecological perspective in the fight against pathogenic microorganisms. This chapter includes additional topics, such as the effect of probiotics and ADP-ribosylation, as well as cAMP, cGMP, and phosphatidylinositol signaling. We also deal here with the problems associated with fecal transplant therapies when using human stool donors that, although asymptomatic, are in fact carriers of bacterial or parasite diseases.

The Case of Lipid II: The Achilles’ Heel of Bacteria

This chapter centers on the crucial role that lipid II plays on the bacterial cell wall and its putative role as the Achilles’ heel of pathogenic bacteria. Lipid II is an essential molecule for bacterial survival and biomolecules targeting this compound could interrupt the bacterial cell wall synthesis, hence causing bacterial lysis. We will concentrate here on several lipid II-acting lantibiotics, such as telavancin, vancomycin, and plusbacin. Additionally, human defensins and moenomycins will be also mentioned, as they constitute putative future biomolecules to fight bacterial infections.

Mechanisms of Drug Efflux and Strategies to Overcome Them as a Way to Control Microbial Growth

The discovery of antibacterials during the twentieth century has been one of the most important events in the history of medicine. The development, production, and use of these new drugs revolutionized clinical practice and industrial microbiology. Certainly, since the beginning of the antibiotic era many lives have been saved. Nevertheless, shortly after the first descriptions of the effects of antibiotics against pathogenic bacteria, the phenomenon of antimicrobial resistance began to be quoted by different researchers. At first, this was not considered a significant clinical event.