Characterization of an aspartate-dependent acid survival system in Yersinia pseudotuberculosis

Enteric bacteria have developed various survival systems that protect against acid stress. In this study, an aspartate-dependent acid survival system is characterized in Yersinia pseudotuberculosis. The expression of aspartase (AspA) was confirmed to be increased at acidic pH by proteomic and lacZ fusion analyses. Addition of aspartate increased acid survival of the wild type but not the aspA knockout mutant. AspA increases acid survival by producing ammonia as demonstrated by mutation and in vitro enzyme activity analyses. This is the first demonstration that an enzyme involved in aspartate metabolism plays a role in acid survival in an enteric bacterium.     

H-NS and H-NS-like proteins in Gram-negative bacteria and their multiple role in the regulation of bacterial metabolism

In Escherichia coli, the H-NS protein plays an important role in the structure and the functioning of bacterial chromosome. A homologous protein has also been identified in several enteric bacteria and in closely related organisms such as Haemophilus influenzae. To get information on their structure and their function, we identified H-NS-like proteins in various microorganisms by different procedures. In silico analysis of their amino acid sequence and/or in vivo experiments provide evidence that more than 20 proteins belong to the same class of regulatory proteins. Moreover, large scale technologies demonstrate that, at least in E. coli, the loss of motility in hns mutants results from a lack of flagellin biosynthesis, due to the in vivo repression of flagellar gene expression. In contrast, several genes involved in adaptation to low pH are strongly induced in a H-NS deficient strain, resulting in an increased resistance to acidic stress. Finally, expression profiling and phenotypic analysis suggest that, unlike H-NS, its paralogous protein StpA does not play any role in these processes.     

Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach (pH 1-3) represents a stressful challenge for enteric pathogenic bacteria, including Escherichia coli, Shigella and Brucella. The hdeA (hns-dependent expression A) gene was found to be crucial for the survival of these enteric bacteria under extremely low pH conditions. We recently demonstrated that HdeA is able to exhibit chaperone-like activity exclusively within the stomach pH range by transforming from a well-folded conformation at higher pH values (above pH 3) into an unfolded conformation at extremely low pH values (below pH 3). This study was performed to characterize the action mechanisms and underlying specific structural features for HdeA to function in this unfolded conformation. In the present study, we demonstrate that the conserved 'amphiphilic' feature of HdeA, i.e. the exposure of the conserved hydrophobic region and highly charged terminal regions, is essential for exhibiting chaperone-like activity under extremely low pH conditions. Mutations that disrupt this amphiphilic feature markedly reduced the chaperone-like activity of HdeA. The results also strongly suggest that this acid-induced chaperone-like activity of HdeA is crucial for acid resistance of the enteric bacteria. Moreover, our new understanding of this amphiphilic structural feature of HdeA helps to better interpret how this unfolded (disordered) conformation could be functionally active.     

Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.     

SPM43.1 Contributes to Acid-Resistance of Non-Symplasmata-Forming Cells in <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> YS19

Pantoea agglomerans YS19 is a rice endophytic bacterium characterized to form multicellular biofilm-like structures called symplasmata. Phenotypic distinctions between symplasmata-forming cells and planktonic cells are crucial for understanding YS19’s survival strategies. In this study, a 43.1 kDa protein SPM43.1 was identified to show significant resistance to the aggregation effect caused by denaturing acidic conditions. MALDI-TOF analysis data indicated that it is a maltose-binding protein homolog while contains sequence homologous to the chaperone protein, ClpB. The purified SPM43.1 protein was detected to exhibit chaperone-like activities at acidic conditions, where its conformation transformed from an ordered to a globally less ordered structure as revealed by circular dichroism spectroscopy, showing a similar property to most chaperone proteins. The expression of SPM43.1 in YS19 is initiated when bacterial cells begin to aggregate, yet its amount in planktonic cells greatly exceeds that in symplasmata-forming cells, suggesting its crucial role to the survival of planktonic cells in experiencing environmental fluctuations. However, the bacterium prefers to form symplasmata, while not to express SPM43.1 proteins, for surviving the artificially set fluctuant (acid here) environments. This study provides valuable information on the life styles and survival strategies of microorganisms that forms multicellular aggregates at specific growth stages.     

Salmonella acid shock proteins are required for the adaptive acid tolerance response.

Salmonella typhimurium, as well as other enteric bacteria, experiences significant fluctuations in H+ ion concentrations during growth in diverse ecological niches. In fact, some pH conditions which should kill cells rapidly, such as stomach acidity, are nevertheless tolerated. The complete mechanism for this tolerance is unknown. However, I have recently demonstrated that S. typhimurium has the ability to survive extreme low pH (pH 3.0 to 4.0) if first adapted to mild pH (pH 5.5 to 6.0). This phenomenon has been referred to as the acidification tolerance response (ATR). The exposure to mild acid is referred to as preshock, and the proteins involved are called preshock ATR proteins. A second type of encounter with acid, called acid shock, involves shifting cells directly from alkaline conditions (pH 7.7) to acid conditions (pH 4.5 or below). During acid shock, the organism immediately ceases reproduction and dramatically changes the expression of at least 52 proteins. All but four are distinct from the preshock ATR proteins. Surprisingly, acid shock alone did not afford significant protection against strong acid challenge in minimal medium. Furthermore, inhibiting protein synthesis prior to acid shock revealed that the acid shock proteins do not appear to contribute to acid survival in minimal medium even at pH 4.3. Constitutive cellular pH homeostatic mechanisms seem sufficient to protect cells at this pH. The data suggest that the induction of acid shock and preshock ATR proteins are separate processes requiring separate signals. However, for S. typhimurium to survive extreme acid conditions, it must induce both the preshock and acid shock systems. Preventing the expression of one or the other eliminates acid tolerance. I propose a two-stage process that allows S. typhimurium to phase in acid tolerance as the environmental pH becomes progressively more acidic.     

Genome Mining and Predictive Functional Profiling of Acidophilic Rhizobacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pt14

Pseudomonas fluorescens Pt14 is a non-pathogenic and acidophilic bacterium isolated from acidic soil (pH 4.65). Genome sequencing of strain Pt14 was performed using Single Molecule Real Time (SMRT) sequencing to get insights into unique existence of this strain in acidic environment. Complete genome sequence of this strain revealed a chromosome of 5,841,722 bp having 5354 CDSs and 88 RNAs. Phylogenomic reconstruction based on 16S rRNA gene, Average Nucleotide Identity (ANI) values and marker proteins revealed that strain Pt14 shared a common clade with P. fluorescens strain A506 and strain SS101. ANI value of strain Pt14 in relation to strain A506 was found 99.23% demonstrating a very close sub-species association at genome level. Further, orthology determination among these three phylogenetic neighbors revealed 4726 core proteins. Functional analysis elucidated significantly higher abundance of sulphur metabolism (>1×) which could be one of the reasons for the survival of strain Pt14 under acidic conditions (pH 4.65). Acidophilic bacteria have capability to oxidize sulphur into sulphuric acid which in turn can make the soil acidic and genome-wide analysis of P. fluorescens Pt14 demonstrated that this strain contributes towards making the soil acidic.     

Functional and structural aspects of Helicobacter pylori acidic stress response factors

Helicobacter pylori is a striking example of adaptation of a bacterium to a very peculiar niche, the human stomach. Despite being a neutralophile, a sophisticated control of gene expression allows it to live and to proliferate in an environment that cycles from nearly neutral to very acidic. Despite the numerous studies performed on the mechanisms of acid adaptation, the physiological function of a large part of the genes products that are up-regulated or down-regulated is often not clear, in particular in the context of the response of the bacterium to an acidic stress. In this review, we discuss the molecular and functional aspects of some of the proteins that are commonly found overexpressed during the acid stress.     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

分子伴侣HdeA与HdeB的作用机制

分子伴侣能够与其他蛋白质的不稳定构象相结合并使其稳定．它的功能之一是能够帮助蛋白质进行正确的折叠与组装．最新研究发现,在肠道致病菌的周质空间中存在着酸性条件下能帮助周质蛋白复性的分子伴侣HdeA和HdeB．HdeA在极端酸性的胃部环境中由二聚体迅速解离成具有伴侣活性的单体,HdeA单体能够和变性的底物蛋白结合防止它们酸诱导聚集,从而保护肠道致病菌安全到达肠道．本文对肠道致病菌的耐酸机制进行了总结,最后对 HdeA和HdeB作用机制的研究近况进行综述,最后对HdeA和HdeB以后的研究方向进行了展望．