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The impact of histidine acid bacterial phytase from Pantoea brenneriAS3 as a feed additive (1000 U/kg feed) on the microbial ecology of the gastrointestinal tract was evaluated. The enzyme was obtained from the culture liquid of a recombinant yeast Pichia pastorispPINK-HC (agpP) by ultrafiltration: a 3.5-fold phytase concentrate with a total activity of about 150,000 units was used as a feed additive. The effect of the enzyme supplementation was assessed by the analysis of the broilers’ caecum microbiota: the number of operational taxonomic units (OTU), the Shannon and Chao-1 indexes were calculated and their values were higher than those of the control group. The microbiota major component analysis graph revealed five distinct clusters, indicating gradual changes in the gut microbial community. The dominant microbial phyla were Bacteroidetes, Firmicutes, and Proteobacteriaand the dominant classes were Bacteroidia, Clostridia, and Negativicutes. Phytase supplementation has led to an increase in the proportion of the Rikenellaceaefamily, represented by Alistipesspp., and the Prevotellaceaefamily, represented by Prevotellaspp. It was concluded that the use of phytase did not have a negative effect on the microbial community and contributed to the change in its qualitative composition, at which the total number of microorganisms and the diversity of the bacterial community increased.

Bacterial intracellular proteinases play a relevant role in coordinating various cellular processes. The genome of the uropathogenic Klebsiella oxytocaNK-1 encodes the gene prtKO, homologous to grimelysin of Serratia grimesii. BLAST analysis showed that a lot of bacteria encode grimelysin-like proteinases, known to be mainly distributed in Enterobacterales.Phylogenetic analysis classified these proteins into 10 clusters. PrtKO was predicted to contain conservative motifs typical for grimelysin using bioinformatic tools. In addition, the structure of metalloproteinase-containing genomic loci is conservative among enterobacteria and consists of the genes involved in global physiological processes. Maximal total intracellular proteolytic activity of K. oxytocaNK-1 is observed during the late stationary phase of growth in LB medium at 37 °C. Limited proteolysis of actin indicates the presence of a grimelysin-like proteinase in bacterial cells during the late stationary growth phase. Hence, we present a first report of a new grimelysin-like intracellular metalloproteinase in K. oxytoca.

Pseudomonas aeruginosais a common device-associated pathogen and also a model object in biofilm research. In this study biofilm growth dynamics of Pseudomonas aeruginosaRSC-2 strain on different culture media was described. It was shown that Mueller-Hinton broth is more suitable for biofilm growth than Lysogeny broth. In vitro biofilm surface was investigated using scanning electron microscopy. Staining with Congo red dye demonstrated that the observed strain can produce amyloid-like fibers that play important role in biofilms and are proposed to be a target for proteinase treatment. The effect of subtilisin-like proteinase of Bacillus pumilus3–19, trypsin, and proteinase K on biofilm was studied. All of these proteinases can destroy biofilms depending on the time of incubation and enzyme activity. After 4 h of incubation, 18–47% of biofilms were destroyed, which can be linked to the resistance of amyloid proteins to proteolytic degradation. However, an increase in the incubation time (to 24 h) led to complete destruction of biofilm.

Morganella morganiiis an opportunistic pathogen which causes a wide range of clinical infections. It is well known that metalloproteases synthesized by pathogenic microorganisms play an important role as virulence factors. In this work, we have conducted bioinformatic analysis, sequencing, and cloning of the gene encoding a putative metalloprotease from M. morganii(AN CP004345.1, MU9_1718). We detected that the amino acid sequence of putative metalloprotease from M. morganiistrain KT has 37% identity to actin-specific metalloproteases grimelysin and protealysin from Serratia grimesiiand Serratia proteamaculans, respectively. Based on ORF MU9_1718 sequence, we have identified a metalloprotease gene in the genome of clinical isolate M. morganiiZM. The gene was cloned under the control of arabinose promoter into pBAD/Myc-His expression vector, and Escherichia coliDH5α cells were subsequently transformed by this vector. We also provide data indicating that the metalloprotease was overexpressed in E. coliDH5α cells as a 35 kDa protein.