Expression of glr gene encoding glutamate racemase in Bacillus licheniformis WX-02 and its regulatory effects on synthesis of poly-γ-glutamic acid

ABSTRACTd-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule ofBacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded byracE1andracE2, are each essential for growth ofB. anthracis, supplyingd-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion ofracE1orracE2did not prevent growth ofB. anthracisSterne (pXO1+pXO2−), the noncapsulating vaccine strain, or ofB. anthracisAmes (pXO1+pXO2+), a fully virulent, capsulating isolate. While mutants with deletions inracE1andracE2were not viable,racE2deletion delayed vegetative growth ofB. anthracisfollowing spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenousd-glutamate. Deletion ofracE1orracE2fromB. anthracisAmes did not affect the production or stereochemical composition of the PDGA capsule. A model is presented wherebyB. anthracis, similar toBacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesized-glutamic acid for peptidoglycan synthesis.IMPORTANCEGlutamate racemases, enzymes that convertl-glutamate tod-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent,B. anthracis, requiresd-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show thatB. anthracispossesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to blockB. anthracisgrowth and achieve therapeutic efficacy.

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Promoting Cell Growth And Poly-Y-Glutamic Acid Production By Boosting The Synthesis of Alanine And D-Alanyl-D-Alanine In Bacillus Licheniformis

10.21203/rs.3.rs-1234457/v1 ◽

2022 ◽

Author(s):

Zheng Zhang ◽

Penghui He ◽

Shiying Hu ◽

Yanqing Yu ◽

Xiaoting Wang ◽

...

Keyword(s):

Cell Growth ◽

Microbial Biomass ◽

Glutamic Acid ◽

Bacillus Licheniformis ◽

Alanine Racemase ◽

High Yield ◽

Effective Strategy ◽

Genes Encoding ◽

Alanine Synthesis ◽

Glutamic Acid Production

Abstract Objective: The production of some bio-chemicals affected by the cell growth. This study aimed at promoting the cell growth by overexpressing the synthesis of peptidoglycans tetrapeptide tail components to improve poly-γ-glutamic acid (γ-PGA) production. Results: L-alanine, D-alanine and D-alanyl-D-alanine are primary precursors for the synthesis of peptidoglycans. The addition of L-alanine and D-alanine significantly increased both the cell growth and production of γ-PGA. Then, several genes encoding key enzymes for L/D-alanine and D-alanyl-D-alanine biosynthesis were overexpressed respectively, including ald (encoding alanine dehydrogenase), dal (encoding alanine racemase) and ddl (encoding D-alanine ligase). The results showed that the overexpression of genes ald , dal and ddl increased the production of γ-PGA by 19.72%, 15.91% and 60.90%, and increased the microbial biomass by 15.58%, 18.34% and 49.85%, respectively. Moreover, we demonstrated that the overexpression of genes ald , dal and ddl increased γ-PGA production mainly by enhancing cell growth rather than providing more precursors. Conclusions: This work illustrated the importance of the L/D-alanine and D-alanyl-D-alanine synthesis to the cell growth and the high yield of γ-PGA, and provided an effective strategy for producing γ-PGA .

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PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

Journal of Bacteriology ◽

10.1128/jb.181.9.2789-2796.1999 ◽

1999 ◽

Vol 181 (9) ◽

pp. 2789-2796 ◽

Cited By ~ 12

Author(s):

Jian Song ◽

Tianhui Xia ◽

Roy A. Jensen

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Phenylalanine Hydroxylase ◽

Transcriptional Level ◽

Gene Encoding ◽

Catalytic Function ◽

Genes Encoding ◽

Catalytic Role ◽

Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

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