This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 μg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 μg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 μg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 μg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Morganella morganii is a Gram-negative bacterial pathogen that causes bacteremia, urinary tract infections, intra-abdominal infections, chorioamnionitis, neonatal sepsis, and newborn meningitis. To control this bacterial pathogen a total of 3565 putative proteins targets in Morganella morganii were screened using comparative subtractive analysis of biochemical pathways annotated by the KEGG that did not share any similarities with human proteins. One of the targets, D-alanyl-D-alanine carboxypeptidase DacB [Morganella] was observed to be implicated in the majority of cell wall synthesis pathways, leading to its selection as a novel pharmacological target. The drug that interacted optimally with the identified target was observed to be Cefoperazone (DB01329) with the estimated free energy of binding -8.9 Kcal/mol. During molecular dynamics simulations; it was observed that DB01328-2exb and DB01329-2exb complexes showed similar values as the control FMX-2exb complex near 0.2 nm with better stability. Furthermore, MMPBSA total free energy calculation showed better binding energy than the control complex for DB01329-2exb interaction i.e. -31.50 (±0.93) kcal/mol. Our presented research suggested that D-alanyl-D-alanine carboxypeptidase DacB could be a therapeutic target and cefoperazone could be a promising ligand to inhibit the D-alanyl-D-alanine carboxypeptidase DacB protein of Morganella morganii. To identify prospective therapeutic and vaccine targets in Morganella morganii, this is the first computational and subtractive genomics investigation of various metabolic pathways exploring other therapeutic targets of Morganella morganii. In vitro/in vivo experimental validation of the identified target D-alanyl-D-alanine carboxypeptidase and the design of its inhibitors is suggested to figure out the best dose, the drug\'s effectiveness, and its toxicity.Communicated by Ramaswamy H. Sarma.

Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain\'s Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50 = 603 CFU) as compared to mice challenged with the parent (LD50 = 1 CFU) or complemented strain (LD50 = 1 CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.