Polyphosphate drives bacterial heterochromatin formation.

Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage­induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.

When the metabolism meets the cell cycle in bacteria.

Nutrients availability is the sinews of the war for single microbial cells, driving growth and cell cycle progression. Therefore, coordinating cellular processes with nutrients availability is crucial, not only to survive upon famine or fluctuating conditions but also to rapidly thrive and colonize plentiful environments. While metabolism is traditionally seen as a set of chemical reactions taking place in cells to extract energy and produce building blocks from available nutrients, numerous connections between metabolic pathways and cell cycle phases have been documented. The few regulatory systems described at the molecular levels show that regulation is mediated either by a second messenger molecule or by a metabolite and/or a metabolic enzyme. In the latter case, a secondary moonlighting regulatory function evolved independently of the primary catalytic function of the enzyme. In this review, we summarize our current understanding of the complex cross-talks between metabolism and cell cycle in bacteria.

Polyphosphate Functions In Vivo as an Iron Chelator and Fenton Reaction Inhibitor.

Maintaining cellular iron homeostasis is critical for organismal survival. Whereas iron depletion negatively affects the many metabolic pathways that depend on the activity of iron-containing enzymes, any excess of iron can cause the rapid formation of highly toxic reactive oxygen species (ROS) through Fenton chemistry. Although several cellular iron chelators have been identified, little is known about if and how organisms can prevent the Fenton reaction. By studying the effects of cisplatin, a commonly used anticancer drug and effective antimicrobial, we discovered that cisplatin elicits severe iron stress and oxidative DNA damage in bacteria. We found that both of these effects are successfully prevented by polyphosphate (polyP), an abundant polymer consisting solely of covalently linked inorganic phosphates. Subsequent in vitro and in vivo studies revealed that polyP provides a crucial iron reservoir under nonstress conditions and effectively complexes free iron and blocks ROS formation during iron stress. These results demonstrate that polyP, a universally conserved biomolecule, plays a hitherto unrecognized role as an iron chelator and an inhibitor of the Fenton reaction.IMPORTANCE How do organisms deal with free iron? On the one hand, iron is an essential metal that plays crucial structural and functional roles in many organisms. On the other hand, free iron is extremely toxic, particularly under aerobic conditions, where iron rapidly undergoes the Fenton reaction and produces highly reactive hydroxyl radicals. Our study now demonstrates that we have discovered one of the first physiologically relevant nonproteinaceous iron chelators and Fenton inhibitors. We found that polyphosphate, a highly conserved and ubiquitous inorganic polyanion, chelates iron and, through its multivalency, prevents the interaction of iron with peroxide and therefore the formation of hydroxyl radicals. We show that polyP provides a crucial iron reservoir for metalloproteins under nonstress conditions and effectively chelates free iron during iron stress. Importantly, polyP is present in all cells and organisms and hence is likely to take on this crucial function in both prokaryotic and eukaryotic cells.

The Chaperone and Redox Properties of CnoX Chaperedoxins Are Tailored to the Proteostatic Needs of Bacterial Species.

Hypochlorous acid (bleach), an oxidizing compound produced by neutrophils, turns the Escherichia coli chaperedoxin CnoX into a powerful holdase protecting its substrates from bleach-induced aggregation. CnoX is well conserved in bacteria, even in non-infectious species unlikely to encounter this oxidant, muddying the role of CnoX in these organisms. Here, we found that CnoX in the non-pathogenic aquatic bacterium Caulobacter crescentus functions as a holdase that efficiently protects 50 proteins from heat-induced aggregation. Remarkably, the chaperone activity of Caulobacter CnoX is constitutive. Like E. coli CnoX, Caulobacter CnoX transfers its substrates to DnaK/J/GrpE and GroEL/ES for refolding, indicating conservation of cooperation with GroEL/ES. Interestingly, Caulobacter CnoX exhibits thioredoxin oxidoreductase activity, by which it controls the redox state of 90 proteins. This function, which E. coli CnoX lacks, is likely welcome in a bacterium poorly equipped with antioxidant defenses. Thus, the redox and chaperone properties of CnoX chaperedoxins were fine-tuned during evolution to adapt these proteins to the specific needs of each species.IMPORTANCE How proteins are protected from stress-induced aggregation is a crucial question in biology and a long-standing mystery. While a long series of landmark studies have provided important contributions to our current understanding of the proteostasis network, key fundamental questions remain unsolved. In this study, we show that the intrinsic features of the chaperedoxin CnoX, a folding factor that combines chaperone and redox protective function, have been tailored during evolution to fit to the specific needs of their host. Whereas Escherichia coli CnoX needs to be activated by bleach, a powerful oxidant produced by our immune system, its counterpart in Caulobacter crescentus, a bacterium living in bleach-free environments, is a constitutive chaperone. In addition, the redox properties of E. coli and C. crescentus CnoX also differ to best contribute to their respective cellular redox homeostasis. This work demonstrates how proteins from the same family have evolved to meet the needs of their hosts.

An essential thioredoxin is involved in the control of the cell cycle in the bacterium Caulobacter crescentus.

Thioredoxins (Trxs) are antioxidant proteins that are conserved among all species. These proteins have been extensively studied and perform reducing reactions on a broad range of substrates. Here, we identified Caulobacter crescentus Trx1 (CCNA_03653; CcTrx1) as an oxidoreductase that is involved in the cell cycle progression of this model bacterium and is required to sustain life. Intriguingly, the abundance of CcTrx1 varies throughout the C. crescentus cell cycle: although the expression of CcTrx1 is induced in stalked cells, right before DNA replication initiation, CcTrx1 is actively degraded by the ClpXP protease in predivisional cells. Importantly, we demonstrated that regulation of the abundance of CcTrx1 is crucial for cell growth and survival as modulating CcTrx1 levels leads to cell death. Finally, we also report a comprehensive biochemical and structural characterization of this unique and essential Trx. The requirement to precisely control the abundance of CcTrx1 for cell survival underlines the importance of redox control for optimal cell cycle progression in C. crescentus.

The anti-inflammatory drug mesalamine targets bacterial polyphosphate accumulation.

Mesalamine serves as the gold standard in treating ulcerative colitis. However, its precise mechanism(s) of action remains unclear. Here, we show that mesalamine treatment rapidly decreases polyphosphate levels in diverse bacteria, including members of the human gut microbiome. This decrease sensitizes bacteria towards oxidative stress, reduces colonization and attenuates persister cell and biofilm formation, suggesting that mesalamine aids in diminishing the capacity of bacteria to persist within chronically inflamed environments.

Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase.

Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.

A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus.

Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability.

The histidine kinase PdhS controls cell cycle progression of the pathogenic alphaproteobacterium Brucella abortus.

Bacterial differentiation is often associated with the asymmetric localization of regulatory proteins, such as histidine kinases. PdhS is an essential and polarly localized histidine kinase in the pathogenic alphaproteobacterium Brucella abortus. After cell division, PdhS is asymmetrically segregated between the two sibling cells, highlighting a differentiation event. However, the function(s) of PdhS in the B. abortus cell cycle remains unknown. We used an original approach, the pentapeptide scanning mutagenesis method, to generate a thermosensitive allele of pdhS. We report that a B. abortus strain carrying this pdhS allele displays growth arrest and an altered DivK-yellow fluorescent protein (YFP) polar localization at the restrictive temperature. Moreover, the production of a nonphosphorylatable PdhS protein or truncated PdhS proteins leads to dominant-negative effects by generating morphological defects consistent with the inhibition of cell division. In addition, we have used a domain mapping approach combined with yeast two-hybrid and fluorescence microscopy methods to better characterize the unusual PdhS sensory domain. We have identified a fragment of the PdhS sensory domain required for protein-protein interaction (amino acids [aa] 210 to 434), a fragment sufficient for polar localization (aa 1 to 434), and a fragment (aa 527 to 661) whose production in B. abortus correlates with the generation of cell shape alterations. The data support a model in which PdhS acts as an essential regulator of cell cycle progression in B. abortus and contribute to a better understanding of the differentiation program inherited by the two sibling cells.