Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock.

Bacteria can form heterogeneous populations containing phenotypic variants of genetically identical cells. The heterogeneity of populations can be considered a bet-hedging strategy allowing adaptation to unknown environmental changes - at least some individual subpopulations or cells might be able to withstand future adverse conditions. Using Percoll gradient centrifugation, we demonstrated that in an Escherichia coli culture exposed to heat shock at 50 °C, two physiologically distinct subpopulations were formed. A high-density subpopulation (HD50) demonstrated continued growth immediately after its transfer to LB medium, whereas the growth of a low-density subpopulation (LD50) was considerably postponed. The LD50 subpopulation contained mainly viable but non-culturable bacteria and exhibited higher tolerance to sublethal concentrations of antibiotics or H2O2 than HD50 cells. The levels of aggregated proteins and main molecular chaperones were comparable in both subpopulations; however, a decreased number of ribosomes and a significant increase in protein oxidation were observed in the LD50 subpopulation as compared with the HD50 subpopulation. Interestingly, under anaerobic heat stress, the formation of the HD50 subpopulation was decreased and culturability of the LD50 subpopulation was significantly increased. In both subpopulations the level of protein aggregates formed under anaerobic and aerobic heat stress was comparable. We concluded that the formation of protein aggregates was independent of oxidative damage induced by heat stress, and that oxidative stress and not protein aggregation limited growth and caused loss of LD50 culturability. Our results indicate that heat stress induces the formation of distinct subpopulations differing in their ability to grow under standard and stress conditions.

Lack of intracellular trehalose affects formation of Escherichia coli persister cells.

Persisters are dormant antibiotic-tolerant cells that usually compose a small fraction of bacterial populations. In this work, we focused on the role of trehalose in persister formation. We found that the ΔotsA mutant, which is unable to synthesize trehalose, produced increased levels of persisters in the early stationary phase and under heat stress conditions. The lack of trehalose in the ΔotsA mutant resulted in oxidative stress, manifested by increased membrane lipid peroxidation after heat shock. Stationary ΔotsA cells additionally exhibited increased levels of oxidized proteins and apurinic/apyrimidinic sites in DNA as compared to WT cells. Oxidative stress caused by the lack of trehalose was accompanied by the overproduction of extracellular indole, a signal molecule that has been shown to stimulate persister formation. Our major conclusion is that intracellular trehalose protects E. coli cells against oxidative stress and limits indole synthesis, which in turn inhibits the formation of persisters.

The formation of persister cells in stationary-phase cultures of Escherichia coli is associated with the aggregation of endogenous proteins.

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.

Antibiotics promoting oxidative stress inhibit formation of Escherichia coli biofilm via indole signalling.

Recent studies have revealed that antibiotics can promote the formation of reactive oxygen species which contribute to cell death. In this study, we report that five different antibiotics known to stimulate production of reactive oxygen species inhibited growth of Escherichia coli biofilm. We demonstrated that supression of biofilm formation was mainly a consequence of the increase in the extracellular concentration of indole, a signal molecule which suppresses growth of bacterial biofilm. Indole production was enhanced under antibiotic-mediated oxidative stress due to overexpression of tryptophanase (TnaA), which catalyzes synthesis of indole. We found that DMSO (dimethyl sulfoxide), a hydrogen peroxide scavenger, or the lack of trypthophanase, which catalyzes production of indole, partly restored formation of E. coli biofilm in the presence of antibiotics. In conclusion, these findings confirmed that antibiotics which promote formation of ROS (reactive oxygen species) can inhibit development of E. coli biofilm in an indole-dependent process.

Small heat shock proteins and protein-misfolding diseases.

Small heat shock proteins (sHsps) are molecular chaperones ubiquitously distributed in numerous species, from bacteria to humans. A conserved C-terminal "alpha-crystallin" domain organized in a beta-sheet sandwich and oligomeric structure are common features of sHsps. sHsps protect cells against many kinds of stresses including heat shock, oxidative and osmotic stress. sHsps recognize unfolded proteins, prevent their irreversible aggregation and facilitate refolding of bound substrates in cooperation with ATP-dependent molecular chaperones (Hsp70/Hsp40). Mammalian sHsps (HSPBs) are multifunctional proteins involved in many cellular processes including those which are not directly related to protein folding and aggregation. HSPBs participate in cell development and cancerogenesis, regulate apoptosis and control cytoskeletal architecture. Recent data revealed that HSPBs also play an important role in membrane stabilization. Mutation in HSPB genes have been identified, which are responsible for the development of cataract, desmin related myopathy and neuropathies. HSPBs are often found as components of protein aggregates associated with protein-misfolding disorders, such as Parkinson's, Alzheimer's, Alexander's and prion diseases. It is supposed that the presence of HSPBs in intra- or extracellular protein deposits is a consequence of the chaperone activity of HSPBs, however more studies are needed to reveal the exact function of HSPBs during the formation (or removal) of disease-related aggregates.

Escherichia coli heat-shock proteins IbpA and IbpB affect biofilm formation by influencing the level of extracellular indole.

The development of Escherichia coli biofilm requires the differential expression of various genes implicated in cell signalling, stress responses, motility and the synthesis of structures responsible for cell attachment. The ibpAB operon is among the stress-response genes most induced during growth of the E. coli biofilm. In this study we demonstrated, to our knowledge for the first time, that the lack of IbpAB proteins in E. coli cells inhibited the formation of biofilm at the air-liquid interface, although it allowed normal planktonic growth. We showed that ibpAB mutant cells experienced endogenous oxidative stress, which might result from a decreased catalase activity. The endogenous oxidative stress in ibpAB cells led to increased expression of tryptophanase, an enzyme which catalyses the synthesis of indole. We demonstrated that the formation of biofilm by the ibpAB mutant was delayed due to the increase in the extracellular concentration of indole, which is known to play the role of a signal molecule, inhibiting biofilm growth.

Role of Escherichia coli heat shock proteins IbpA and IbpB in protection of alcohol dehydrogenase AdhE against heat inactivation in the presence of oxygen.

Escherichia coli small heat shock proteins IbpA and IbpB are molecular chaperones that bind denatured proteins and facilitate their subsequent refolding by the ATP-dependent chaperones DnaK/DnaJ/GrpE and ClpB. In vivo, the lack of IbpA and IbpB proteins results in increased protein aggregation under severe heat stress or delayed removal of aggregated proteins at recovery temperatures. In this report we followed the appearance and removal of aggregated alcohol dehydrogenase, AdhE, in E. coli submitted to heat stress in the presence of oxygen. During prolonged incubation of cells at 50 degrees C, when AdhE was progressively inactivated, we initially observed aggregation of AdhE and thereafter removal of aggregated AdhE. In contrast to previous studies, the lack of IbpA and IbpB did not influence the formation and removal of AdhE aggregates. However, in DeltaibpAB cells AdhE was inactivated and oxidized faster than in wild type strain. Our results demonstrate that IbpA and IbpB protected AdhE against thermal and oxidative inactivation, providing that the enzyme remained soluble. IbpA and IbpB were dispensable for the processing of irreversibly damaged and aggregated AdhE.

Aggregation of Escherichia coli proteins during stationary phase depends on glucose and oxygen availability.

In natural environments, bacteria are often challenged by nutrient starvation and other stresses. As a consequence, cell growth is arrested and bacteria enter stationary phase. In this report, we demonstrate that during stationary phase, Escherichia coli cells accumulate aggregates of misfolded proteins and complexes of Dps (starvation-induced protein) with chromosomal DNA. We found that the formation of multicomponent protein aggregates and insoluble Dps-DNA complexes depended on growth conditions and was influenced by the availability of oxygen and glucose in a medium. Aerobic stationary cells grown in unbuffered medium supplemented with glucose contained insoluble Dps-DNA, whereas multicomponent protein aggregates were accumulated under glucose starvation. On the contrary, under oxygen depletion, Dps-DNA complexes were formed in the absence of glucose, whereas multicomponent protein aggregates appeared in the presence of glucose. The mechanisms responsible for this phenomenon remain to be elucidated; however, we demonstrated that in MOPS-buffered cultures the level of insoluble Dps and protein aggregates was decreased.

Escherichia coli heat-shock proteins IbpA/B are involved in resistance to oxidative stress induced by copper.

The small heat-shock proteins IbpA/B are molecular chaperones that bind denatured proteins and facilitate their subsequent refolding by the ATP-dependent chaperones DnaK, DnaJ, GrpE and ClpB. In this report, we demonstrate that IbpA/B participate in the defence against copper-induced stress under aerobic conditions. In the presence of oxygen, DeltaibpA/B cells exhibit increased sensitivity to copper ions and accumulate elevated amounts of oxidized proteins, while under oxygen depletion, the DeltaibpA/B mutation has no effect on copper tolerance. This indicates that IbpA/B protect Escherichia coli cells from oxidative damage caused by copper. We show that AdhE, one of the proteins exposed to oxidation, is protected by IbpA/B against copper-mediated inactivation both in vivo and in vitro.

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells.

Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

The Escherichia coli small heat-shock proteins IbpA and IbpB prevent the aggregation of endogenous proteins denatured in vivo during extreme heat shock.

The roles of the Escherichia coli IbpA and IbpB chaperones in protection of heat-denatured proteins against irreversible aggregation in vivo were investigated. Overproduction of IbpA and IbpB resulted in stabilization of the denatured and reversibly aggregated proteins (the S fraction), which could be isolated from E. coli cells by sucrose gradient centrifugation. This finding is in agreement with the present model of the small heat-shock proteins' function, based mainly on in vitro studies. Deletion of the ibpAB operon resulted in almost twofold increase in protein aggregation and in inactivation of an enzyme (fructose-1,6-biphosphate aldolase) in cells incubated at 50 degrees C for 4 h, decreased efficiency of the removal of protein aggregates formed during prolonged incubation at 50 degrees C and affected cell viability at this temperature. IbpA/B proteins were not needed for removal of protein aggregates or for the enzyme protection/renaturation in cells heat shocked at 50 degrees C for 15 min. These results show that the IbpA/B proteins are required upon an extreme, long-term heat shock. Overproduction of IbpA but not IbpB caused an increase of the level of beta-lactamase precursor, which was localized in the S fraction, together with the IbpA protein, which suggests that the unfolded precursor binds to IbpA but not to IbpB. Although in the wild-type cells both E. coli small heat-shock proteins are known to localize in the S fraction, only 2% of total IbpB co-localized with the aggregated proteins in the absence of IbpA, while in the absence of IbpB, the majority of IbpA was present in the aggregates fraction.