HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria

The X-ray crystal structure of the Escherichia coli stress response protein HDEA has been determined at 2.0 A resolution. The single domain alpha-helical protein is found in the periplasmic space, where it supports an acid resistance phenotype essential for infectivity of enteric bacterial pathogens, such as Shigella and E. coli. Functional studies demonstrate that HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins. We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions.     

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.     

Multidrug resistance in lactic acid bacteria: molecular mechanisms and clinical relevance

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by t he same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.     

Long-term shifts in patterns of antibiotic resistance in enteric bacteria

Several mechanisms are responsible for the ability of microorganisms to tolerate antibiotics, and the incidence of resistance to these compounds within bacterial species has increased since the commercial use of antibiotics became widespread. To establish the extent of and changes in the diversity of antibiotic resistance patterns in natural populations, we determined the MICs of five antibiotics for collections of enteric bacteria isolated from diverse hosts and geographic locations and during periods before and after commercial application of antibiotics began. All of the pre-antibiotic era strains were susceptible to high levels of these antibiotics, whereas 20% of strains from contemporary populations of Escherichia coli and Salmonella enterica displayed high-level resistance to at least one of the antibiotics. In addition to the increase in the frequency of high-level resistance, background levels, conferred by genes providing nonspecific low-level resistance to multiple antibiotics, were significantly higher among contemporary strains. Changes in the incidence and levels of antibiotic resistance are not confined to particular segments of the bacterial population and reflect responses to the increased exposure of bacteria to antimicrobial compounds over the past several decades.     

Copper resistance mechanisms in bacteria and fungi

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced effiux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.     

Phage resistance in lactic acid bacteria

The interactions between lactic acid bacteria and their phages are commercially significant. Current research has focused on the elucidation of the mechanisms and genetics of phage resistance. Phage resistance genes have been linked to plasmid DNA for Streptococcus lactis and Streptococcus cremoris, and preliminary studies suggest the operation of mechanisms such as the prevention of phage adsorption, restriction/modification, and abortive infection. Some phage resistance plasmids can be conjugally transferred, providing a means of dissemination among phage-sensitive strains for the construction of phage-resistant starter cultures.     

细菌锌离子抗性机制研究进展

锌(Zn)是生命代谢中重要的微量金属元素,但锌过量会对细胞造成毒害作用。细菌通过一些特有的机制来解除重金属离子对它们的毒害。该文重点介绍了细菌对高浓度Zn2+的抗性机制,主要包括外排机制(RND蛋白家族、CDF蛋白家族和P-型ATPase)、螯合机制和外排后结合机制。通过这些机制细菌能有效控制胞内Zn2+浓度,保护其不受过量Zn2+的毒害,但抗性机制往往不依赖单一的抗性系统,而是多种系统协调作用的结果。细菌Zn2+抗性机制的研究将有助于进一步揭示生物是如何应对高浓度金属离子的胁迫及相应的适应性规律。     

Detergent-shock response in enteric bacteria

Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.     

Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria

Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined. APH(3')-I and AAC(3)-II were shown to be the most frequent. The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E. coli clinical strains. DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments. The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization. Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.     

Plasmid-mediated high-level gentamicin resistance among enteric bacteria isolated from pet turtles in Louisiana

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 microg/ml) to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria, such as Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2,000 microg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes aac(3)-IIa and aac(3)-VIa; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of Salmonella spp. from turtle eggs.     

A propionate-inducible expression system for enteric bacteria

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P(prpB) system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P(prpB) of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P(prpB) system exhibited negligible basal expression by addition of glucose to the medium.     

Molecular basis of active copper resistance mechanisms in Gram-negative bacteria

Copper is a metallic element that is crucial for cell metabolism; however, in extended concentrations, it is toxic for all living organisms. The dual nature of copper has forced organisms, including bacteria, to keep a tight hold on cellular copper content. This challenge has led to the evolution of complex mechanisms that on one hand enable them to deliver the essential element and on the other to protect cells against its toxicity. Such mechanisms have been found in both eukaryotic and prokaryotic cells. In bacteria a number of different systems such as extra- and intracellular sequestration, enzymatic detoxification, and metal removal from the cell enabling them to survive in the presence of high concentration of copper have been identified. Gram-negative bacteria, due to their additional compartment, need to deal with both cytoplasmic and periplasmic copper. Therefore, these bacteria have evolved intricate and precisely regulated systems which interact with each other. In this review the active mechanisms of copper resistance at their molecular level are discussed.     

Bacteriophage and bacteriophage resistance in lactic acid bacteria

Abstract: The study of bacteriophage-host interactions has been instrumental in the development of genetic systems in many genera, and laid many of the foundations of modern molecular genetics. Research into bacteriophage and bacteriophage resistance in the lactic acid bacteria has moved into a new and exciting dimension in recent years. Mechanisms such as adsorption inhibition, restriction and modification, and abortive infection which have been detected and described phenotypically over the past decade are now being subjected to molecular analysis, and this has led to a better understanding of the nature and variety of resistance systems employed by lactic acid bacteria to combat phage attack. In addition, analysis of different bacteriophage has increased our knowledge of these ubiquitous particles to the point where it is possible to construct novel phage resistances based on the phage genome itself. This review outlines the recent progress in the molecular analysis of bacteriophage, bacteriophage resistance and counter resistance, and the construction of novel resistance mechanisms.     

Survival of enteric bacteria in seawater

Enteric bacteria exposed to the marine environment simultaneously encounter a variety of abiotic and biotic challenges. Among the former, light appears to be critical in affecting seawater survival; previous growth history plays a major part in preadaptation of the cells, and stationary phase cells are generally more resistant than exponentially growing ones. Predation, mostly by protozoa, is probably the most significant biotic factor. Using Escherichia coli as a model, a surprisingly small number of genes was found that, when mutated, significantly affect seawater sensitivity of this bacterium. Most prominent among those is rpoS, which was also dominant among genes induced upon transfer to seawater.