Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis

Zymomonas mobilis ( Z. mobilis ) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs ( ZMO0197 , ZMO0959 , ZMO1089 ) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis .

Functional insights into the high-molecular-mass penicillin-binding proteins of Streptococcus agalactiae revealed by gene deletion and transposon mutagenesis analysis

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus , or GBS) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four non-essential PBPs is poorly characterized in GBS. We deleted the pbp1a , pbp1b , pbp2a , and pbp2b genes individually from a genetically well-characterized serotype V GBS strain, and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain (i) none of the pbp isogenic mutant strains had a significant growth defect in THY rich medium, (ii) isogenic mutant strains Δ pbp1a and Δ pbp1b had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant strains Δ pbp1a and Δ pbp2b had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined genes essential for the viability of wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM-PBPs) are enzymes required for bacterial cell-wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM-PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM-PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell-wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.

Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.

Hydrocortisone decreases lethality and inflammatory cytokine and nitric oxide production in rats challenged with B. anthracis cell wall peptidoglycan

Background Lethal B. anthracis infection produces high proinflammatory peptidoglycan (PGN) burdens in hosts. We investigated whether the lethality and inflammation anthrax PGN can produce are related. Methods At 6 h before and the start of 24 h anthrax PGN infusions, rats (n = 198) were treated with diluent (controls) or one of three IV-doses of either hydrocortisone (125, 12.5 or 1.25 mg/kg) or TNF-soluble receptor (TNFsr; 2000, 1000 or 333 μg/kg), non-selective and selective anti-inflammatory agents, respectively. Results Compared to controls, hydrocortisone 125 and 12.5 mg/kg each decreased 7-day lethality (p ≤ 0.004). Hydrocortisone 125 mg/kg decreased IL-1β, IL-6, TNFα, MCP, MIP-1α, MIP-2, RANTES and nitric oxide (NO) blood levels at 4 and 24 h after starting PGN (except MCP at 24 h). Each decrease was significant at 4 h (except MIP-1α that was significant at 24 h) (p ≤ 0.05). Similarly, hydrocortisone 12.5 mg/kg decreased each measure at 4, 24 and 48 h (except TNFα at 24 h and MIP-1α at 24 and 48 h and NO at 48 h). Decreases were significant for IL-6 and NO at 4 h and RANTES at 48 h (p ≤ 0.05). Hydrocortisone 1.25 mg/kg had non-significant effects. Each TNFsr dose decreased lethality but non-significantly. However, when doses were analyzed together, TNFsr decreased lethality in a potential trend (p = 0.16) and IL-6 and NO significantly at 4 h (p = 0.05). Conclusions Peptidoglycan-stimulated host inflammation may contribute to B. anthracis lethality.

Influence of Carbohydrate Residues on Antibacterial Activity of Vancomycin

This paper presents synthesis of vancomycin derivatives modified with selected 1- and 2-aminoalditols to carboxylic function and 2,5-anhydro-D-mannose and D-talose to amino function of vancosamine via reductive alkylation. MIC and MBC of these derivatives were determined for reference strains of bacteria: Staphylococcus aureus ATCC 25923, ATCC 6538, ATCC 6538/P, S. epidemidis ATCC 14490, E. faecium PCM 1859, E. faecalis PCM 2673, S. pyogenes PCM 465, and S. pneumonia ATCC 49619 and compared with the activity of vancomycin and its aglycone. Our findings confirm that sugar fragments can play an important role in the mechanism of interaction of vancomycin with bacterial cell wall peptidoglycan.

Investigating β-lactam drug targets in Mycobacterium tuberculosis using chemical probes

Tuberculosis is a deadly disease that requires a flexible arsenal of drugs to treat it. Although β-lactam antibiotics are rarely used to treat Mycobacterium tuberculosis (Mtb) infections today, the targets of these drugs are present in the bacterium. Moreover, the cell wall peptidoglycan of Mtb contains an abundance of unusual (3→3) cross-links. These cross-links are formed by enzymes called L,D-transpeptidases, which are susceptible to inhibition by the carbapenem class of antibiotics. We developed new small molecule probes to investigate the L,D-transpeptidases and other β-lactam drug targets in Mtb. We synthesized probes based on three classes of antibiotics, a monobactam, cephalosporin, and carbapenem. For the carbapenem, we synthesized a meropenem analogue conjugated to a far-red fluorophore. This probe was particularly useful in identifying active L,D-transpeptidases in protein gel-resolved lysates. Next we analyzed β-lactam targets in lysates from both hypoxic and actively-replicating cultures of Mtb. We identified numerous targets, including transpeptidases, carboxypeptidases, and the β-lactamase BlaC. Overall, we provide evidence that Mtb dynamically regulates the enzymes responsible for maintaining cell wall peptidoglycan and that meropenem is a good inhibitor of those enzymes.