Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically, and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with Vmax of 3.9 ± 0.2 μmol/min/mg and K m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN-PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN-PscL heterotrimeric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN-PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN-PscL complex in T3SS mediated pathogenic pathway.

The silkworm forms cocoon to protect its pupa that survives for months inside the cocoon without being affected by various environmental stresses. To understand the possible mode of pupal survival within the cocoon encasement, we investigate the cause that protects the cocoon. During the end of the spinning process, we have isolated different bacterial species from the cocoon surface. These are identified using molecular techniques and checked for their abilities to form biofilm in vitro. The bacteria are able to form biofilm either individually or in consortia. Of which, Bacillus and Erwinia species are prominent biofilm formers. Interestingly, these bacteria have the ability to form biofilm on the cocoon mimetic surface of the silk protein Sericin Hope that contains only sericin. The origin and the behavior of the bacteria lead us to hypothesize the possible role of biofilm layer on the cocoon surface, which provides protection from adverse environmental conditions.

Type Three Secretion (T3S) ATPases are involved in delivery of virulent factors from bacteria to their hosts (through injectisome) in an energy (ATP) dependent manner during pathogenesis. The activities of these ATPases are tightly controlled by their specific regulators. In Yersinia enterocolitica, YsaN was predicted as a putative ATPase of the Ysa-Ysp Type Three Secretion System (T3SS) based on sequence similarity with other T3S ATPases. However detailed study and characterization of YsaN and its regulation remains largely obscure. Here, in this study, we have successfully cloned, over-expressed, purified and characterized the molecular properties of YsaN from Yersinia enterocolitica. YsaN acts as a Mg(2+) dependent ATPase and exists in solution as higher order oligomer (dodecamer). The ATPase activity of oligomeric YsaN is several fold higher than the monomeric form. Furthermore, by employing in silico studies we have identified the existence of a negative regulator of YsaN--a hypothetical protein YE3555 (termed 'YsaL'). To verify the functionality of YsaL, we have evaluated the biochemical and biophysical properties of YsaL. Purified YsaL is dimeric in solution and strongly associates with YsaN to form a stable heterotrimeric YsaL-YsaN complex (stoichiometry--2∶1). The N terminal 6-20 residues of YsaN are invariably required for stable YsaL-YsaN complex formation. YsaL inhibited the ATPase activity of YsaN with a maximum inhibition at the molar ratio 2∶1 (YsaL: YsaN). In short, our studies provide an insight into the presence of YsaN ATPase in Yersinia enterocolitica and its regulator YsaL. Our studies also correlate the functionality of one of the existing protein interaction networks that possibly is indispensable for the energy dependent process of Ysa-Ysp T3SS in pathogenic Yersinia enterocolitica.