Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.

The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functional properties. Up to now, the high price makes this application nearly impossible. In this work, the increased expression for Escherichia coli optimized Lys-C was investigated. The cloned sequence had a short artificial N-terminal pro-peptide (MGSK). The expression of MGSK-Lys-C was tested using three expression vectors and five E. coli host strains. The highest expression rate was obtained for the expression system consisting of the host strain E. coli JM109 and the rhamnose inducible expression vector pJOE. A Lys-C activity of 9340 ± 555 nkatTos-GPK-pNA/Lculture could be achieved under optimized cultivation conditions after chemical refolding. Furthermore, the influence of the native pre-N-pro peptide of Lys-C from Lysobacter enzymogenes ssp. enzymogenes ATCC 27796 on Lys-C refolding was investigated. The pre-N-pro peptide was expressed recombinantly in E. coli JM109 using the pJOE expression vector. The optimal concentration of the pre-N-pro peptide in the refolding procedure was 100 µg/mLrefolding buffer and the Lys-C activity could be increased to 541,720 nkatTos-GPK-pNA/Lculture. With the results presented, the expensive lysyl endopeptidase can be produced in high activity and high amounts and the potential of Lys-C for tailor-made protein hydrolysates with bioactive (e.g. antihypertensive) and/or techno functional (e.g. foaming, emulsifying) properties can be investigated in future time studies.

Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF.

Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

[Detection of AlpA and AlpB lytic endopeptidase propeptides of Lysobacter sp. XL1 by sandwich-enzyme immunoassay based on monoclonal antibodies].

The extracellular lytic endopeptidases AlpA and AlpB of the bacterium Lysobacter sp. XL1 are highly homologous and synthesized as precursors consisting of signal peptide, propeptide and mature form. In this work, two monoclonal antibodies against propeptide endopeptidase AlpA (ProA) and eleven against propeptide endopeptidase AlpB (ProB) were obtained to study the AlpA and AlpB endopeptidases secretion. The affinity constants of the antibodies against ProA were 2.9 x 10(9) and 3.5 x 10(9) M(-1), and the affinity constants of the antibodies against ProB were from 1.5 x 10(8) to 2.2 x 10(9) M(-1). The obtained antibodies did not have cross-reactivity between themselves, as well as mature forms of the enzymes. The monoclonal antibodies based sandwich-type enzyme immunoassay has been developed for measuring the propeptide in a native form. The linear range of determination ProA was 1.5-100 ng/mL with 6% error of measurement, and for determining ProB 0.2-6.25 ng/mL with 6% error. Using the developed assay, ProA and ProB propeptides have been detected in cell lysates of Lysobacter sp. XL1 in an amount 1.18 ± 0.03 ng and 0.096 ± 0.002 ng per 1 OD540 of the bacterial culture, respectively. The immunochemical assay for detection various forms of AlpA and AlpB lytic endopeptidases can be useful when dealing with issues related to their secretion into the environment.

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage.

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants. Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38). Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity. The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.

A novel laccase with urate oxidation activity from Lysobacter sp. T-15.

A unique urate-oxidizing enzyme was identified in a bacterium, strain T-15. Based on its phylogenetic, physiological and biochemical properties, strain T-15 was deemed to be a novel species within the genus Lysobacter. The enzyme expressed in Lysobacter sp. T-15 was composed of 592 amino acids and contained four consensus copper-binding sites, and the recombinant enzyme was, at least in this study, speculated to have three Cu ions per subunit. The primary structure of the enzyme was 33% identical to Marinomonas mediterranea polyphenol oxidase, but it showed no significant similarity to any known urate oxidase. With urate as the substrate, the catalytic efficiency (k(cat)/K(m)) of recombinant enzyme was 4.0 × 10(2) s(-)(1)mM(-)(1), and it was not inhibited by xanthine, a strong urate oxidase inhibitor. The enzyme also showed activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), 2,6-dimethoxyphenol and bilirubin, with catalytic efficiencies of 4.9 × 10(2), 1.1 × 10(2) and 3.6 × 10(3) s(-)(1)mM(-)(1), respectively. We deemed the enzyme would be a member of laccase from its broad substrate specificity. However, typical laccase and other multi-copper oxidases such as bilirubin oxidase and ascorbate oxidase seldom exhibit urate oxidation activity. These results would expand the laccase substrate range to include urate.

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374.

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.