Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum

Panicker, Indu S.; Kanci, Anna; Markham, Philip F.; Browning, Glenn F.

doi:10.1099/mic.0.000327

Cited by 3 publications

(1 citation statement)

References 36 publications

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“…In P. aeruginosa Lpp that are located in the inner membrane have Gly+2 followed by Asp/Glu +3 (Remans, Vercammen, Bodilis, & Cornelis, 2010). Recently, it has been shown that aa variations in +2 of the alkaline phosphatase (PhoA) expressed in Mycoplasma gallisepticum did not affect the retention of PhoA to the membrane (Panicker, Kanci, Markham, & Browning, 2016). Finally, in Bacillus subtilis it was found that Gly+2 facilitates release of Lpp while a Ser+2 favors withholding in the membrane (Tjalsma & van Dijl, 2005).…”

Section: Discussionmentioning

confidence: 99%

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane

2017

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In gram‐negative bacteria, the ABC transporter LolCDE complex translocates outer membrane‐specific lipoproteins (Lpp) from the inner membrane to the outer membrane. Lpp possessing aspartate (Asp) at position +2 are not translocated because it functions as a LolCDE avoidance signal. In gram‐positive bacteria, lacking an outer membrane and the Lol system, Lpp are only anchored at the outer leaflet of the cytoplasmic membrane. However, the release of Lpp particularly in pathogenic or commensal species is crucial for immune modulation. Here, we provide evidence that in Staphylococcus aureus Asp at position +2 plays a role in withholding Lpp to the cytoplasmic membrane. Screening of published exoproteomic data of S. aureus revealed that Lpp mainly with Gly or Ser at position +2 were found in exoproteome, but there was no Lpp with Asp+2. The occurrence of Lpp with Asp+2 is infrequent in gram‐positive bacteria. In S. aureus USA300 only seven of the 67 Lpp possess Asp+2; among them five Lpp represented Lpl lipoproteins involved in host cell invasion. Our study demonstrated that replacing the Asp+2 present in Lpl8 with a Ser enhances its release into the supernatant. However, there is no different release of Asp+2 and Ser+2 in mprF mutant that lacks the positive charge of lysyl‐phosphatidylglycerol (Lys‐PG). Moreover, substitution of Ser+2 by Asp in SitC (MntC) did not lead to a decreased release indicating that in staphylococci positions +3 and +4 might also be important for a tighter anchoring of Lpp. Here, we show that Asp in position +2 and adjacent amino acids contribute in tightening the anchoring of Lpp by interaction of the negative charged Asp with the positive charged Lys‐PG.

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Section: Discussionmentioning

confidence: 99%

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane

2017

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Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

Mascelli,

Garcia,

Gardner

2024

Appl Environ Microbiol

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Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α−1,2), nigerose (α−1,3), and isomaltose (α−1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in the amy13E gene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis of amy13E found that this gene was essential for kojibiose and isomaltose metabolism in C. japonicus . Interestingly, a Δ amy13E mutant was not deficient for cyclodextrin or pullulan utilization in C. japonicus ; however, heterologous expression of the gene in E. coli was sufficient for cyclodextrin-dependent growth. Biochemical analyses found that Cj Amy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of the Cj Amy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications. IMPORTANCE Understanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

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Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

Garcia

Narrett

2020

Appl Environ Microbiol

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The α-diglucoside trehalose has historically been known as a component of bacterial stress response, though more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate active enzymes, specifically those of the glycoside hydrolase family 37, to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate active enzymes predicted to have the same biochemical activity, and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus. This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus. Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an E. coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose, and is an acidic α-α-trehalase. Bioinformatic and mutational analysis indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus, and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. IMPORTANCE The metabolism of α-diglucosides like trehalose is becoming increasingly important due to its inclusion in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. The results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

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Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum

Cited by 3 publications

References 36 publications

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation

Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

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