Effect of temperature, pH and plasmids on in vitro biofilm formation in Escherichia coli

Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37°C or 25°C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different ability of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25°C, with biofilm being hardly detectable at 37°C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37°C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated

The yhiM gene codes for an inner membrane protein involved in GABA export in Escherichia coli

In order to survive the exposure to acid pH, Escherichia coli activates molecular circuits leading from acid tolerance to extreme acid resistance (AR). The activation of the different circuits involves several global and specific regulators affecting the expression of membrane, periplasmic and cytosolic proteins acting at different levels to dampen the harmful consequences of the uncontrolled entry of protons intracellularly. Many genes coding for the structural components of the AR circuits (protecting from pH ≤ 2.5) and their specific transcriptional regulators cluster in a genomic region named AFI (acid fitness island) and respond in the same way to global regulators (such as RpoS and H-NS) as well as to anaerobiosis, alkaline, cold and respiratory stresses, in addition to the acid stress. Notably some genes coding for structural components of AR, though similarly regulated, are non-AFI localised. Amongst these the gadBC operon, coding for the major structural components of the glutamate-based AR system, and the ybaS gene, coding for a glutaminase required for the glutamine-based AR system. The yhiM gene, a non-AFI gene, appears to belong to this group. We mapped the transcription start of the 1.1 kb monocistronic yhiM transcript: it is an adenine residue located 22 nt upstream a GTG start codon. By real-time PCR we show that GadE and GadX equally affect the expression of yhiM under oxidative growth conditions. While YhiM is partially involved in the RpoS-dependent AR, we failed to detect a significant involvement in the glutamate- or glutamine-dependent AR at pH ≤ 2.5. However, when grown in EG at pH 5.0, the yhiM mutant displays impaired GABA export, whereas when YhiM is overexpressed, an increases of GABA export in EG medium in the pH range 2.5–5.5 is observed. Our data suggest that YhiM is a GABA transporter with a physiological role more relevant at mildly acidic pH, but not a key component of AR at pH < 2.5

Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

The Glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH =2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis

Clonaggio e produzione di forme mutanti di glutammato decarbossilasi di Escherichia coli capaci di catalizzare la reazione di decarbossilazione in un ampio intervallo di pH

La presente invenzione concerne mutanti di glutammato decarbossilasi di Escherichia coli capaci di catalizzare la reazione di decarbossilazione in un ampio intervallo di pH, pertanto vantaggiosamente utilizzabili per rimuovere e/o misurare il glutammato in un campione, produrre GABA, monitorare il pH e come biosensori

Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: Function, distribution and biomedical implications of the gadBC operon

For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate-dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP-dependent enzyme which performs a proton-consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamatein/g-aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH-dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non-pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co-regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA-enriched foods possess health-promoting properties. © 2012 Blackwell Publishing Ltd

Glutamate decarboxylase in bacteria

gamma -Aminobutyrate (GABA) is a non-proteinaceous amino acid which plays different roles in the living systems. GABA is biochemically produced by the irreversible a-decarboxylation of l-glutamate catalysed by glutamate decarboxylase (Gad), a widespread enzyme distributed among eukaryotes and prokaryotes. Structural features of the plant, mammalian and bacterial Gads are reported in the literature. In pathogenic bacteria, Gad activity has been linked to protection from acid stress, but the possibility cannot be excluded that it might perform other physiological roles. Indeed, the screening of lactic acid bacteria (LAB) based on their capacity to synthesize GABA opens a new perspective for the production of GABA containing dairy foods. The aim of this chapter is to provide an overview of the best characterized Gad enzymes in pathogenic and non-pathogenic bacteria, along with a description of the potential use of Gad as a source of GABA in functional foods and in biotechnological products

Biochemical characterization of a variant of Escherichia coli glutamate decarboxylase with improved GABA production at alkaline pH.

The Federation of European Microbiological Societies (FEMS) and the Spanish Society for Microbiology (SEM) have joined​ forces to give you the best of microbiology. Come to the modern and friendly Mediterranean city Valencia, meet fellow microbiologists and update your knowledge with the state of the art on antimicrobial resistance and infections, food microbiology, sustainability, climate change and many more topics. Key disciplines including molecular approaches, biodiversity, bioremediation, eukaryotic microbes, virology and others will be examined in order to advance our understanding of current and future challenges.​​

Analoghi e derivati di amminoacidi dicarbossilici come antibatterici

Questa invenzione si colloca nell’ambito scientifico della lotta alle resistenze multiple sviluppate dai batteri verso gli antibiotici attualmente in commercio. L’invenzione stravolge il concetto che gli antibiotici possano avere solo una certa natura chimica, intorno alla quale si è sviluppato un mercato che non riesce più ad essere innovativo, e che ha prodotto molecole chimicamente imparentate tra loro. Con questa invenzione inoltre, si vuole portare all’attenzione di soggetti interessati l’efficacia di molecole che agiscono sul metabolismo centrale nei batteri, e per le quali allo stato attuale e non ci sono indicazioni di tossicità sugli animali che ne precluderebbero l’impiego sistemico/topico