Biology subjects

Hamm, C. W.

Publications and source records attributed to Hamm, C. W..

3 recordsLinked to original sources

bioRxiv2026

The roles and synthesis of inorganic polyphosphate in Bacillus cereus

Inorganic polyphosphate (polyP) is a universally conserved biopolymer central to bacterial stress survival, yet understanding of its roles derives almost entirely from Gram-negative models in which polyP accumulates intracellularly following nutrient downshift. We examined polyP metabolism in the Gram-positive spore-forming bacterium Bacillus cereus using deletions of the polyP kinases PPK1 and PPK2 and the exopolyphosphatase PPX. Intracellular polyP synthesis required PPK1 and was opposed by PPX and PPK2: ppx mutants accumulated polyP in sporulation medium by 24 hours, ppx ppk2 double mutants accumulated more, and no ppk1 mutant accumulated any. A ppk1 ppx double mutant could not be generated, suggesting that unopposed PPK2 activity is lethal. Unlike Escherichia coli and Pseudomonas aeruginosa, B. cereus did not accumulate polyP after shift to minimal medium, increasing only modestly in stationary phase. Fluorescence and transmission electron microscopy localized intracellular polyP to electron-dense granules within ribosome-depleted cytoplasm. Cells bearing these granules remained membrane-intact yet failed to resume growth over 8 hours in rich medium, leading us to propose that polyP drives ribosome sequestration into condensates and a hibernation-like state. Unexpectedly, B. cereus also released close to 100{micro}M polyP extracellularly during late stationary phase, even in a ppk1 ppk2 mutant lacking both known synthetases. Extracellular polyP resisted hydrolysis by purified PPX even after deproteinization, indicating an atypical structure. Bacillus thuringiensis and Bacillus anthracis released similar amounts of extracellular polyP. Together these results identify two distinct polyP pools in the B. cereus group: a PPK1-dependent intracellular pool and an extracellular pool made by an uncharacterized pathway. ImportanceBacillus cereus is a spore-forming bacterium that causes foodborne illness and persists in soil and food-processing environments, where survival depends on managing phosphate and energy reserves during starvation. Inorganic polyphosphate (polyP), an ancient polymer used by nearly all cells to withstand stress, has been studied almost entirely as a molecule stored inside bacteria. We show that Bacillus cereus maintains two separate polyP pools. The internal pool is made by a known enzyme (PPK1) and is associated with dormant cells whose protein-making machinery appears to be packed away. The external pool is made without any known polyP-synthesizing enzyme, pointing to a novel polyP synthesis pathway that is shared with the close relatives Bacillus thuringiensis and Bacillus anthracis.

microbiology↗

bioRxiv2025

Probiotic biogeography and sepsis prevention in the neonatal intestine

Neonatal infection is one of the leading causes of neonatal morbidity and mortality worldwide, particularly in those born prematurely or with low birth weight. Probiotic bacteria have been demonstrated to protect against the development of neonatal intestinal dysbiosis and are widely used in peri- and post-natal clinical settings. However, formulations and efficacy are highly variable, highlighting a critical gap in the current understanding of the mechanistic underpinnings of successful probiotic interventions in this population. Furthermore, current studies on probiotic efficacy largely rely on indirect or relative readouts of intestinal bacterial burden. Herein, we directly mapped the biogeography of intestinal colonization and quantify the probiotic effects of Escherichia coli Nissle 1917 (EcN) and Ligilactobacillus murinus strain V10 against Klebsiella pneumoniae dysbiosis across the span of the neonatal murine intestine. Despite substantial differences in biogeography within the intestine, both EcN and L. murinus V10 significantly reduced K. pneumoniae colonization and mortality from K. pneumoniae sepsis, with EcN doing so much more robustly. EcNs probiotic effect was partially dependent on its ability to respire oxygen. Contrary to the dominant paradigm and practice in the probiotic field, combining multiple probiotic strains did not necessarily increase efficacy. Simultaneous treatment with EcN and L. murinus V10 was less effective than EcN treatment alone at preventing death from sepsis. These results highlight important variables which must be taken into account in the design of effective future probiotic intervention strategies. IMPORTANCEIn this work we use a mouse model of late-onset neonatal sepsis (LOS) to rigorously test fundamental assumptions that underlie the current paradigm for understanding the impact of probiotics on intestinal disease. We demonstrate that two distantly related probiotic bacteria (Escherichia coli Nissle 1917 and Ligilactobacillus murinus V10) can each effectively reduce both intestinal colonization and death caused by the LOS pathobiont Klebsiella pneumoniae, acting by distinct ecological and molecular mechanisms. Our results provide new evidence that will be critical for designing and implementing safe and effective probiotic treatment regimens for LOS, a devastating and difficult to treat disease. More broadly, our results show that ecological principles are key to understanding how interventions that modulate the gut microbiome work, and that some of the assumptions underlying current interventions need to be reevaluated, especially when it comes to combining multiple probiotic strains and species.

microbiology↗

bioRxiv2024

Inorganic polyphosphate and the stringent response coordinately control cell division and cell morphology in Escherichia coli

Bacteria encounter numerous stressors in their constantly changing environments and have evolved many methods to deal with stressors quickly and effectively. One well known and broadly conserved stress response in bacteria is the stringent response, mediated by the alarmone (p)ppGpp. (p)ppGpp is produced in response to amino acid starvation and other nutrient limitations and stresses and regulates both the activity of proteins and expression of genes. Escherichia coli also makes inorganic polyphosphate (polyP), an ancient molecule evolutionary conserved across most bacteria and other cells, in response to a variety of stress conditions, including amino acid starvation. PolyP can act as an energy and phosphate storage pool, metal chelator, regulatory signal, and chaperone, among other functions. Here we report that E. coli lacking both (p)ppGpp and polyP have a complex phenotype indicating previously unknown overlapping roles for (p)ppGpp and polyP in regulating cell division, cell morphology, and metabolism. Disruption of either (p)ppGpp or polyP synthesis led to formation of filamentous cells, but simultaneous disruption of both pathways resulted in cells with heterogenous cell morphologies, including highly branched cells, severely mislocalized Z-rings, and cells containing substantial void spaces. These mutants also failed to grow when nutrients were limited, even when amino acids were added. These results provide new insights into the relationship between polyP synthesis and the stringent response in bacteria and point towards their having a joint role in controlling metabolism, cell division, and cell growth. IMPORTANCECell division is a fundamental biological process, and the mechanisms that control it in Escherichia coli have been the subject of intense research scrutiny for many decades. Similarly, both the (p)ppGpp-dependent stringent response and inorganic polyphosphate (polyP) synthesis are well-studied, evolutionarily ancient, and widely conserved pathways in diverse bacteria. Our results indicate that these systems, normally studied as stress-response mechanisms, play a coordinated and novel role in regulating cell division, morphology, and metabolism even under non-stress conditions.

microbiology↗