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(1) Background: d-alanine-d-alanine ligase (DdlA), an effective target for drug development to combat against Mycobacterium tuberculosis (Mtb), which threatens human health globally, supplies a substrate of d-alanyl-d-alanine for peptidoglycan crosslinking by catalyzing the dimerization of two d-alanines. To obtain a better understanding of DdlA profiles and develop a colorimetric assay for high-throughput inhibitor screening, we focused on explicating and characterizing Tb-DdlA. (2) Methods and Results: Rv2981c (ddlA) was expressed in Escherichia coli, and the purified Tb-DdlA was identified using (anti)-polyhistidine antibody followed by DdlA activity confirmation by measuring the released orthophosphate via colorimetric assay and the yielded d-alanyl-d-alanine through high performance thin layer chromatography (HP-TLC). The kinetic assays on Tb-DdlA indicated that Tb-DdlA exhibited a higher affinity to ATP (KmATP: 50.327 ± 4.652 μmol/L) than alanine (KmAla: 1.011 ± 0.094 mmol/L). A colorimetric assay for Tb-DdlA activity was developed for high-throughput screening of DdlA inhibitors in this study. In addition, we presented an analysis on Tb-DdlA interaction partners by pull-down assay and MS/MS. Eight putative interaction partners of Tb-DdlA were identified. (3) Conclusions: Our dataset provided a valuable resource for exploring Tb-DdlA biology, and developed an easy colorimetric assay for screening of Tb-DdlA inhibitors.

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. Highlights To achieve the highest lysozyme production, the genes were expressed in E. coli BL21. Genes, Lyz-1 (N-acetylmuramoyl-L-alanine amidase) and Lyz-2 (D-alanyl-D-alanine carboxypeptidase), were introduced. Lyz1 showed the highest antimicrobial activity, whereas Lyz2 had the lowest. The disintegrating effect of bacteria and yeast has been indicated by AFM analysis. Lyz1 can cleave amide bond between glycan and peptide components of the peptidoglycan. Graphical abstract

can cause different nosocomial infections, especially urinary tract infection (UTI). Pathogenicity of is driven by various virulence factors; however, no specific genetic pattern is restricted to a particular type of infection. The current study aimed to investigate the correlation between different virulence factors in clinical isolates causing UTIs. We phenotypically analyzed 60 urinary isolates, identified as , for biofilm formation, gelatinase, protease and hemolytic activities by Crystal Violet assay, gelatin hydrolysis, casein hydrolysis and blood agar hemolysis assays, respectively. Additionally, we detected different genes associated with species identification, virulence phenotypes, adherence and quorum sensing by the polymerase chain reaction (PCR). The detected genes included D-alanine-D-alanine ligase ( ), cytolysin ( ), gelatinase ( ), serine protease ( ), faecal streptococci regulator locus genes ( ), pili ( ), adhesin to collagen of ( ) and aggregation substance ( ). All isolates formed biofilms, mostly with strong to moderate ability. Although was detected in 87% of the isolates, only 22% of the isolates had gelatinase activity. Similar phenotype-genotype incongruities were observed with hemolysis and casein hydrolysis activities, as the isolates that expressed these two phenotypes were fewer than those carrying the genes encoding them. A clear variability in virulence gene distribution among the isolates was observed, and no particular pattern was associated with UTI. Whereas all isolates carried at least and , whose products are involved in adherence, which is a virulence phenotype that is required for urinary colonization, six isolates carried the entire set of investigated genes. Statistical analysis of the results suggests as a biomarker for hemolytic activity, as a diagnostic biomarker for the gelatinase activity, and as predictors for biofilm formation strength in .

Stenotrophomonas maltophilia is a multidrug resistant pathogen associated with high mortality and morbidity in patients having compromised immunity. The efflux systems of S . maltophilia include SmeABC and SmeDEF proteins, which assist in acquisition of multiple-drug-resistance. In this study, proteome based mapping was utilized to find out the potential drug targets for S . maltophilia strain k279a. Various tools of computational biology were applied to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. The CD-HIT analysis selected 4315 proteins from total proteome count of 4365 proteins. Geptop identified 407 essential proteins, while the BlastP revealed approximately 85 non-homologous proteins in the human genome. Moreover, metabolic pathway and subcellular location analysis were performed for essential bacterial genes, to describe their role in various cellular processes. Only two essential proteins (Acyl-[acyl-carrier-protein]—UDP-N acetyl glucosamine O-acyltransferase and D-alanine-D-alanine ligase) as candidate for potent targets were found in proteome of the pathogen, in order to design new drugs. An online tool, Swiss model was employed to model the 3D structures of both target proteins. A library of 5000 phytochemicals was docked against those proteins through the molecular operating environment (MOE). That resulted in to eight inhibitors for both proteins i.e. enterodiol, aloin, ononin and rhinacanthinF for the Acyl-[acyl-carrier-protein]—UDP-N acetyl glucosamine O-acyltransferase, and rhazin, alkannin beta, aloesin and ancistrocladine for the D-alanine-D-alanine ligase. Finally the ADMET was done through ADMETsar. This study supported the development of natural as well as cost-effective drugs against S . maltophilia . These inhibitors displayed the effective binding interactions and safe drug profiles. However, further in vivo and in vitro validation experiment might be performed to check their drug effectiveness, biocompatibility and their role as effective inhibitors.

Background The deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia , including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea. Results Significant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia . Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD + homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems. Conclusions We identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.

The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) threatens global health. The mechanism of vancomycin resistance of VRSA without vanA gene acquisition was not fully elucidated. Therefore, we aimed to determine the mechanism of vancomycin resistance of VRSA besides that by vanA gene acquisition. In this study, we obtained vancomycin-resistant strains (V036-V64; MIC = 64 µg /ml) from susceptible strain (V036; MIC = 0.5 µg /ml) by exposure of vancomycin in vitro and examined the phenotypic characteristics and antibiotic susceptibility profiles of the resistant strain (V036-V64). To identify the genetic variations caused vancomycin resistance, we determined the complete genome sequences of V036 and V036-V64 and analyzed for single-nucleotide polymorphisms (SNPs) between two strains. Morphologically, V036-V64 had a twofold thicker cell wall compared with V036. Linezolid, rifampicin, and ceftaroline had similar MIC ranges against V036-V64 and V036, but V036-V64 showed lower susceptibilities to daptomycin and telavancin. We detected eight single-nucleotide polymorphisms differing between V036-V64 and V036: rimM (G16D), ssaA2 (G128A), rpsK (P60R), rpoB (R917C), walK (T492R), d-alanyl-d-alanine carboxypeptidase (L307I), vraT (A152V), and chromosome segregation ATPase (T440I). This study demonstrates that, under selective pressure, by the accumulation of mutations in genes related to cell wall synthesis, vancomycin-susceptible S. aureus can develop thicker cell walls and, hence, develop high vancomycin resistance. Thus, we highlight a novel vanA-negative mechanism for VRSA emergence.

Streptococcus pneumoniae (S. pneumoniae) is an important pathogen worldwide that causes pneumococcal infections which are related to high rates of morbidity and mortality especially in young children, older adults, and immune-compromised persons. Antibiotic resistance in S. pneumoniae is a serious problem across the world from time to time, resulting in treatment failure and diminished value of older medicines. Therefore, the objective of this study was to identify new putative drug targets against S. pneumoniae serotype 23F by using subtractive genomics. By using bioinformatics tools such as NCBI, UniProt KB, PDB, KEGG, DEG, PSORTb, CD hit, DrugBank database, and other softwares, proteins involved in unique metabolic pathways of S. pneumoniae serotype 23F were studied. The result indicates that this serotype consists of 97 metabolic pathways of which 74 are common with that of human, and 23 pathways are unique to the serotype 23F. After investigation and analysis of essentiality, nonhomology, subcellular localization, having drug targets, and enzymatic activity, four proteins were prioritized as druggable targets. These druggable proteins include UDP-N-acetylglucosamine 1-carboxyvinyltransferase, UDP-N-acetyl muramate dehydrogenase, D-alanine-D-alanine ligase, and alanine racemase that are found in S. pneumoniae serotype 23F. All these four proteins are essential, are nonhomologous with human proteins, have drug targets, and are located in cell cytoplasm. Therefore, the authors recommend these proteins to be used for efficient drug design against S. pneumoniae serotype 23F after experimental validation for essentiality and druggability.

Metabolic engineering is a substantial approach for escalating the production of biochemical products. Cell biomass is lowered by system constraints and toxication carried on by the aggregation of metabolites that serve as inhibitors of product synthesis. In order to increase the production of biochemical products, it is important to trace the relationship between alanine metabolism and biomass. According to our investigation, the appropriate concentration of additional L/D-alanine (0.1 g/L) raised the cell biomass (OD600) in Bacillus licheniformis in contrast to the control strain. Remarkably, it was also determined that high levels of intracellular L/D-alanine and D-alanyl-D-alanine were induced by the overexpression of the ald, dal, and ddl genes to accelerate cell proliferation. Our findings clearly revealed that 0.2 g/L of L-alanine and D-alanine substantially elevated the titer of poly-γ-glutamic acid (γ-PGA) by 14.89% and 6.19%, correspondingly. And the levels of γ-PGA titer were hastened by the overexpression of the ald, dal, and ddl genes by 19.72%, 15.91%, and 16.64%, respectively. Furthermore, overexpression of ald, dal, and ddl genes decreased the by-products (acetoin, 2,3-butanediol, acetic acid and lactic acid) formation by about 14.10%, 8.77%, and 8.84% for augmenting the γ-PGA production. Our results also demonstrated that overexpression of ald gene amplified the production of lichenysin, pulcherrimin and nattokinase by about 18.71%, 19.82% and 21.49%, respectively. This work delineated the importance of the L/D-alanine and D-alanyl-D-alanine synthesis to the cell growth and the high production of bio-products, and provided an effective strategy for producing bio-products.

Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production.

Antibiotic resistance poses an increasing threat to global health. To tackle this problem, the identification of principal reservoirs of antibiotic resistance genes (ARGs) plus an understanding of drivers for their evolutionary selection are important. During a PCR-based screen of ARGs associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a d -alanine- d -alanine ligase ( ddl6 and ddl7 ) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to d -cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6 . A SNP was found to be responsible for this difference in resistance phenotype between the two ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and d -cycloserine binding pockets of Ddl.