Biology subjects

Hernandez, S. B.

Publications and source records attributed to Hernandez, S. B..

5 recordsLinked to original sources

bioRxiv2022

Genome-wide peptidoglycan profiling of Vibrio cholerae

Most bacteria cells are protected by a peptidoglycan cell wall. Defining the chemical structure of the peptidoglycan has been instrumental to characterize cell wall associated proteins and to illuminate the mode of action of cell wall-acting antibiotics. However, a major roadblock for a comprehensive understanding of peptidoglycan homeostasis has been the lack of methods to conduct large-scale, systematic studies. Here we have developed and applied an innovative high throughput peptidoglycan analytical pipeline to analyze the entire non-essential, arrayed mutant library of Vibrio cholerae. The unprecedented breadth of these analyses revealed that peptidoglycan homeostasis is preserved by a large percentage of the genome organized in complex networks that functionally link peptidoglycan features with genetic determinants. As an example, we discovered a novel bifunctional penicillin-binding protein in V. cholerae. Collectively, genome-wide peptidoglycan profiling provides a fast, easy, and unbiased method for systematic identification of the genetic determinants of peptidoglycan synthesis and remodeling.

microbiology↗

bioRxiv2020

L-arabinose induces the formation of viable non-proliferating Vibrio cholerae spheroplasts

A general survival strategy of many life forms faced with harmful growth conditions is to enter into a non-proliferating state until conditions suitable for growth are restored. In bacteria, this survival strategy is associated with antimicrobial tolerance, chronic infections and environmental dispersion. In particular, the agent of the deadly human disease cholera, Vibrio cholerae, undergoes a morphological transition from a rod-shaped proliferative form to a spherical non-proliferating form after exposure to cold or cell wall targeting antibiotics. Growth is resumed when the adverse conditions have ceased. Here, we show that a component of the hemicellulose and pectin of terrestrial plants, L-arabinose, triggers the formation of non-proliferating V. cholerae spherical cells, which are able to return to growth when L-arabinose is removed from the growth medium. We found that the cell wall of L-arabinose treated V. cholerae cells has a peptidoglycan composition similar to the cell wall of spheroplasts and that they revert to a wild-type morphology through the formation of branched cells like L-forms. Unlike L-forms, however, they are osmo-resistant. Through a random Tn genetic screen for mutants insensitive to L-arabinose, we identified genes involved in the uptake and catabolism of galactose and in glycolysis. We hypothesize that L-arabinose is enzymatically processed by the galactose catabolism and glycolysis pathways until it is transformed in a product that cannot be further recognized by V. cholerae enzymes. Accumulation of this enzymatic by-product triggers the formation of viable non-dividing cell wall deficient spherical cells.

microbiology↗

bioRxiv2019

Modulation of peptidoglycan synthesis by recycled cell wall tetrapeptides

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized by the cell, where they are recycled for synthesis of new PG. Although the PG recycling pathway is widely conserved, its components are not essential and its roles in cell wall homeostasis are not well-understood. Here, we identified LdcV, a Vibrio cholerae L,D-carboxypeptidase that cleaves the terminal D-Alanine from recycled murotetrapeptides. In the absence of ldcV, recycled tetrapeptides accumulated in the cytosol, leading to two toxic consequences for the cell wall. First, incorporation of tetrapeptide-containing PG precursors into the cell wall led to reduction in D,D-cross-linkage between stem peptides, diminishing PG integrity. Second, tetrapeptide accumulation led to a decrease in canonical UDP-pentapeptide precursors, reducing PG synthesis. Thus, LdcV and the recycling pathway promote optimal cell wall assembly and composition. Furthermore, Ldc substrate preference for murotetrapeptides containing canonical (D-Alanine) vs. non-canonical (D-Methionine) D-amino acids is conserved, suggesting that accumulation of tetrapeptide recycling intermediates may modulate PG homeostasis in environments enriched in non-canonical-muropeptides via substrate competition.

microbiology↗

bioRxiv2019

The cell division factor ZapB is required for bile resistance in Salmonella enterica

A gene annotated as yiiU in the genome of Salmonella enterica serovar Typhimurium encodes a protein homologous to E. coli ZapB, a non-essential cell division factor involved in Z-ring assembly. ZapB- null mutants of S. enterica are bile-sensitive. The ZapB protein is degraded in the presence of sodium deoxycholate (DOC), and degradation appears to involve the Lon protease. The amount of zapB mRNA increases in the presence of a sublethal concentration of DOC. This increase is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. DOC-induced increase of the zapB transcript is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. Increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thus providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.\n\nIMPORTANCEBile salts have bactericidal activity as a consequence of membrane disruption, protein denaturation and DNA damage. However, intestinal bacteria are resistant to bile. Envelope structures such as the lipopolysaccharide and the enterobacterial common antigen act as barriers that reduce intake of bile salts. Remodelling of the outer membrane and the peptidoglycan, activation of efflux pumps, and upregulation of stress responses also contribute to bile resistance. This study adds the cell division factor ZapB (and presumably the Z-ring) to the list of cellular functions involved in bile resistance.

microbiology↗

bioRxiv2019

Muropeptides Stimulate Growth Resumption from Stationary Phase in Escherichia coli

When nutrients run out, bacteria enter a dormant metabolic state. This low or undetectable metabolic activity helps bacteria to preserve their scant reserves for future, but also diminishes their ability to trace the environment for new growth-promoting substrates. However, neighboring microbial growth is a sure indicator of favorable environment and thus, can serve as a cue for exiting the dormancy. Here we report that for Escherichia coli this cue is the basic peptidoglycan unit (i.e. muropeptide). We show that several forms of muropeptides can stimulate growth resumption of dormant E. coli cells, but the sugar - peptide bond is crucial for activity. We also demonstrate that muropeptides from several different species can induce growth resumption of E. coli and also Pseudomonas aeruginosa. These results, together with the previous identification of muropeptides as germination signal for bacterial spores, makes muropeptides rather universal cue for bacterial growth.

microbiology↗