Biofilm and motility in response to environmental and host-related signals in Gram negative opportunistic pathogens.

Most bacteria can switch between a planktonic, sometimes motile, form and a biofilm mode, in which bacterial cells can aggregate and attach to a solid surface. The transition between these two forms represents an example of bacterial adaptation to environmental signals and stresses. In 'environmental pathogens', namely, environmental bacteria that are also able to cause disease in animals and humans, signals associated either with the host or with the external environment, such as temperature, oxygen availability, nutrient concentrations etc., play a major role in triggering the switch between the motile and the biofilm mode, via complex regulatory mechanisms that control flagellar synthesis and motility, and production of adhesion factors. In this review article, we present examples of how environmental signals can impact biofilm formation and cell motility in the Gram negative bacteria Pseudomonas aeruginosa, Escherichia coli and in the Burkholderia genus, and how the switch between motile and biofilm mode can be an essential part of a more general process of adaptation either to the host or to the external environment.

High-throughput screening of a collection of known pharmacologically active small compounds for inhibitors of Salmonella invasion and intracellular replication.

AIMS: The aim of this study was to screen a chemical library consisting of over 1200 pharmacologically active, already approved off-patent compounds, to determine whether any of the compounds reduced or eliminated the invasion or intracellular replication phenotypes of Salmonella enterica serovar Typhimurium (S. Typhimurium). METHODS AND RESULTS: LacZ reporter and tissue culture-based infection assays were used to screen for compounds that significantly reduced expression of key virulence genes, and were required for the invasion or intracellular replication phenotypes of S. Typhimurium in host cells. The search lead to the discovery of four compounds that reduced invasion by between 90-100%, and two compounds that reduced intracellular replication by between 65-93% at concentrations of either 2, 10 or 50 µg ml-1 , relative to an untreated control strain. CONCLUSIONS: We identified six compounds that significantly reduced expression of S. Typhimurium virulence genes resulting in decreased in vitro virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: The emergence of multidrug-resistant strains of Salmonella poses a considerable and growing worldwide threat to human and animal health. The screening of off-patent chemical libraries represents a potential discovery route for novel antimicrobials.

Effects of sublethal doses of silver nanoparticles on Bacillus subtilis planktonic and sessile cells.

AIMS: Due to their antimicrobial activity, silver nanoparticles (Ag-NPs) are being increasingly used in a number of industrial products. The accumulation of Ag-NPs in the soil might affect plant growth-promoting rhizobacteria and, in turn, the plants. We describe the effects of Ag-NPs on the soil bacteria Azotobacter vinelandii and Bacillus subtilis. METHODS AND RESULTS: In growth-inhibition studies, A. vinelandii showed extreme sensitivity to Ag-NPs, compared to B. subtilis. We investigated the effects of Ag-NPs at subinhibitory concentrations, both on planktonic and sessile B. subtilis cells. As determined by 2,7-dichlorofluorescein-diacetate assays, Ag-NPs increase the formation of reactive oxygen species in planktonic cells, but not in sessile cells, suggesting the activation of scavenging systems in biofilms. Consistently, proteomic analysis in B. subtilis Ag-NPs-treated biofilms showed increased production of proteins related to quorum sensing and involved in stress responses and redox sensing. Extracellular polysaccharides production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to stress. CONCLUSIONS: At low concentrations, Ag-NPs killed A. vinelandii and affected cellular processes in planktonic and sessile B. subtilis cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Re-direction of gene expression, linked to selective toxicity, suggests a strong impact of Ag-NPs on soil bacterial communities.

Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene.

The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.

Transcriptional responses to DNA damage.

In Escherichia coli, DNA repair and protective responses are regulated at the transcriptional level. Regulatory mechanisms have evolved that allow cells to respond to DNA damage by mounting the appropriate responses. The regulatory proteins controlling these responses are activated when they recognize the presence of a specific DNA damaging agent, the production of specific DNA lesions, or the production of damage intermediates resulting from replication of lesions containing DNA. Transcription of the responses to DNA damage are induced when the activated regulatory proteins stimulate transcription of the genes they control by a variety of complex and unique molecular mechanisms.

Activation and repression of transcription initiation in bacteria.

Transcription initiation is the principal step at which bacterial gene expression is regulated. Bacterial transcription is due to a single multisubunit RNA polymerase. The potential transcription initiation rate of any promoter is set by the efficiency with which RNA polymerase recognizes the different promoter sequence elements. The sigma subunit plays the major role in the process of promoter recognition. Different RNA polymerase sigma subunits can guide RNA polymerase to different promoters. The E. coli genome encodes seven different sigma subunits, each of which allows the cell to respond to different environmental stimuli. A large number of transcription factors up-regulate and down-regulate expression from different promoters in response to environmental signals. Many transcription activators function by making a direct interaction with RNA polymerase. Some activators function by altering the conformation of promoter DNA. Most transcription repressors function by blocking access of RNA polymerase to their target promoter. In some cases, optimal repression depends on multiply bound repressor molecules that interact in complex ways. Many promoters are regulated by more than one transcription factor. A variety of mechanisms whereby a promoter can be regulated by a repressor and an activator, or by two activators, is known.

Artificial nutrition and bone marrow transplantation.

Parenteral nutrition has a central role in the supportive therapy of patients submitted to a BMT. A central catheter is mandatory for transfusions, antibiotic therapy and a proper nutrition. A good nutritional support contributes to maintain hydration, reduce lean body mass loss, increase patient comfort and improve survival in patients who can not eat or absorb for a prolonged period of time. After a BMT metabolic complications are frequent and require careful monitoring; in critical care patients, the major risks are electrolyte and glucose disturbances. Liver disease is a main metabolic complication of PN, but it can occur in any cancer patient due to therapy or to graft-versus-host disease. Its best prevention requires the avoidance of prolonged enteral fasting.

Expression of the Escherichia coli ada regulon in stationary phase: evidence for rpoS-dependent negative regulation of alkA transcription.

The Escherichia coli Ada protein activates sigma(70)-dependent transcription at three different promoters (ada, aidB, and alkA) in response to alkylation damage of DNA. During stationary phase, however, the methylated form of Ada shuts off expression of alkA; this repression is specific for sigma(S)-dependent transcription. Thus, at the alkA promoter, the Ada protein can act as both a positive and negative modulator of the adaptive response to alkylation damage, depending on the cell's physiological state.

The Escherichia coli Ada protein can interact with two distinct determinants in the sigma70 subunit of RNA polymerase according to promoter architecture: identification of the target of Ada activation at the alkA promoter.

The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada, aidB, and alkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit sigma70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in sigma70 region 4 results in decreased Ada-dependent binding of RNA polymerase to the alkA promoter in vitro and impairs alkA transcription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-sigma70 interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307-13312, 1998), we showed that a set of negatively charged amino acids in sigma70 region 4 is involved in meAda-sigma70 interaction at the ada and aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affect meAda-dependent transcription at ada and aidB. Unlike the sigma70 amino acids involved in the interaction with meAda at the ada and aidB promoters, K593, K597, and R603 are not conserved in sigmaS, an alternative sigma subunit of RNA polymerase mainly expressed during the stationary phase of growth. While meAda is able to promote transcription by the sigmaS form of RNA polymerase (EsigmaS) at ada and aidB, it fails to do so at alkA. We propose that meAda can activate transcription at different promoters by contacting distinct determinants in sigma70 region 4 in a manner dependent on the location of the Ada binding site.

Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters.

The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613). Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription. Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on meAda-sigma70 interaction.

The RNA polymerase alpha subunit carboxyl-terminal domain is required for both basal and activated transcription from the alkA promoter.

Expression of the Escherichia coli adaptive response genes (ada, aidB, and alkA) is regulated by the transcriptional activator, Ada. However, the interactions of RNA polymerase and Ada with these promoters differ. In this report we characterize the interactions of Ada, methylated Ada (meAda), and RNA polymerase at the alkA promoter and contrast these interactions with those characterized previously for the ada and aidB promoters. At the alkA promoter, we do not detect the RNA polymerase alpha subunit-mediated binary complex detected at the ada and aidB promoters. In the presence of either of these two activators, RNA polymerase protects the alkA core promoter, including the elements at -35 and -10, and is more efficient in transcription initiation in vitro. RNA polymerase holoenzyme containing the alpha subunit mutation R265A is severely impaired in Ada-independent basal alkA transcription, shows no activation by Ada or meAda, and fails to bind the alkA promoter in vitro. Binding of the purified wild type alpha subunit to alkA was not detected, but a complex of promoter DNA, Ada or meAda, and alpha was observed in gel shift assays. These observations suggest that both forms of Ada protein activate alkA transcription by enhancing RNA polymerase holoenzyme and alpha subunit binding.

Antimicrobial activities of chemically modified thiazolyl peptide antibiotic MDL 62,879 (GE2270A).

MDL 62,879 (GE2270A) 1 is a new inhibitor of elongation factor-Tu (EF-Tu) and belongs to the class of thiazolyl peptide antibiotics. Controlled acid hydrolysis of 1 followed by treatment with base resulted in the lost of the two terminal amino acids and in the formation of water-soluble MDL 62,935 2. Although less active in vitro than its parent compound, 2 was able to inhibit by 50% an Escherichia coli cell-free protein synthesis system at roughly the same concentration of 1. MDL 62,935 2 was subjected to further modification at the beta-phenylserine residue. Derivatives obtained from 2 were less active in both antimicrobial (MIC) and enzymatic (IC50) assays. This suggests that beta-phenylserine plays an important role for the inhibition of EF-Tu by 1 and 2.

Antibiotics MDL 62,879 and kirromycin bind to distinct and independent sites of elongation factor Tu (EF-Tu).

Antibiotic MDL 62,879 inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). In this study we show that the inhibition of protein synthesis by MDL 62,879 in an Escherichia coli cell-free system was fully reversed by addition of stoichiometric amounts of EF-Tu but not by large excesses of EF-Ts, ribosomes, or aa-tRNA. MDL 62,879 bound tightly to EF-Tu and formed a stable 1:1 MDL 62,879:EF-Tu (M:EF-Tu) complex. We show that binding of MDL 62,879 to EF-Tu strongly affects the interaction of EF-Tu with aa-tRNA and causes rapid dissociation of preformed EF-Tu.aa-tRNA complex, suggesting that inhibition of aa-tRNA binding is due to a conformational change in EF-Tu rather than competition for the aa-tRNA binding site. Indication of a conformational change in EF-Tu induced by MDL 62,879 is further confirmed by proteolytic cleavage experiments: MDL 62,879 binding strongly protects EF-Tu against trypsin cleavage. The observed effects of MDL 62,879 appear to be different from those of the kirromycin class of antibiotics, which also inhibit protein synthesis by binding to EF-Tu, suggesting two distinct binding sites. Indeed, the M:EF-Tu complex was able to bind stoichiometric amounts of kirromycin to form a 1:1:1 M:EF-Tu:kirromycin (M:EF-Tu:K) complex, providing direct evidence that the two antibiotics bind to independent and distinct sites on the EF-Tu molecule. The interaction of the M:EF-Tu:K complex with aa-tRNA and other co-factors suggest that the contemporary binding of the two antibiotics locks EF-Tu into an intermediate conformation in which neither antibiotic exhibits complete dominance.

The leucine-responsive regulatory protein (Lrp) acts as a specific repressor for sigma s-dependent transcription of the Escherichia coli aidB gene.

The product of the Escherichia coli aidB gene is homologous to human isovaleryl-coenzyme A dehydrogenase (IVD), an enzyme involved in the breakdown of the amino acid leucine. The aidB gene is not expressed constitutively, but its transcription is induced via distinct mechanisms in response to: (i) exposure to alkylating agents; (ii) acetate at a slightly acidic pH; and (iii) anoxia. Induction by alkylating agents is mediated by the transcriptional activator Ada, in its methylated form (meAda); the other forms of induction are Ada independent and require sigma s, the alternative sigma factor mainly expressed during the stationary phase of bacterial growth. In this report we show that, in the absence of any transcriptional factor, aidB is efficiently transcribed in vitro by the sigma s, but not by the sigma 70, form of RNA polymerase holoenzyme. In the presence of meAda, levels of transcription by both forms of RNA polymerase are significantly increased. However, sigma s-dependent transcription of aidB is inhibited both in vitro and in vivo by binding of the transcriptional regulator Lrp (leucine responsive protein) to the aidB promoter region (PaidB). Lrp acts as a specific repressor for sigma s-dependent transcription of aidB. Leucine counteracts Lrp binding to P aidB, as does binding to P aidB of me Ada, which causes Lrp to dissociate from the promoter. The physiological significance of aidB transcription regulation is discussed.

RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes.

The ada and aidB genes are part of the adaptive response to DNA methylation damage in Escherichia coli. Transcription of the ada and the aidB genes is triggered by binding of the methylated Ada protein (meAda) to a specific sequence located 40-60 base pairs upstream of the transcriptional start, which is internal to an A/T-rich region. In this report we demonstrate that the Ada binding site is also a binding site for RNA polymerase. RNA polymerase is able to bind the -40 to -60 region of the ada and the aidB promoters in the absence of meAda, and its binding is mediated by the alpha subunit. This region resembles the UP element of the rrnB P1 promoter in location, sequence and mechanism of interaction with RNA polymerase. We discuss the function of UP-like elements in positively controlled promoters and provide evidence that Ada does not act by enhancing RNA polymerase binding affinity to the promoter region. Instead, Ada stimulates transcription by modifying the nature of the RNA polymerase-promoter interaction, allowing RNA polymerase to recognize the core promoter -35 and -10 elements in addition to the UP-like element.

Antibiotic GE37468 A: a new inhibitor of bacterial protein synthesis. I. Isolation and characterization.

GE37468 A is a new thiazolyl peptide antibiotic obtained by fermentation of Streptomyces sp. strain ATCC 55365. It inhibits bacterial protein synthesis by acting on elongation factor Tu and is structurally and functionally related to the GE2270 class of EF-Tu inhibitors. It is active in vitro against Gram-positive bacteria and Bacteroides fragilis, and protects mice against Staphylococcus aureus infection.

Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein. Evidence for a new consensus sequence for Ada-binding sites.

The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage. Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69. Through DNaseI protection assays, we show that methylated Ada (meAda) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint. Binding of meAda is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of meAda. Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidB in vitro transcription. The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E. coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis. Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for meAda might be AATnnnnnnG-CAA. Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site. Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site. We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.

Structure and transcriptional regulation of the Escherichia coli adaptive response gene aidB.

Expression of the Escherichia coli aidB gene is induced in vivo by alkylation damage in an ada-dependent pathway and by anaerobiosis or by acetate at pH 6.5 in an ada-independent fashion. In this report, we present data on aidB gene structure, function, and regulation. The aidB gene encodes a protein of ca. 60 kDa that is homologous to several mammalian acyl coenzyme A dehydrogenases. Accordingly, crude extracts from an aidB-overexpressing strain showed isovaleryl coenzyme A dehydrogenase activity. aidB overexpression also reduced N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis. Both ada- and acetate/pH-dependent induction of aidB are regulated at the transcriptional level, and the same transcriptional start point is used for both kinds of induction. Ada protein plays a direct role in aidB regulation: methylated Ada is able to bind to the aidB promoter region and to activate transcription from aidB in an in vitro transcription-translation system using crude E. coli extracts.

Sensitivity of elongation factor Tu (EF-Tu) from different bacterial species to the antibiotics efrotomycin, pulvomycin and MDL 62879.

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.