RESUMO
BACKGROUND: It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low. RESULTS: We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10-6/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results. CONCLUSION: This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.
Assuntos
Escherichia coli/fisiologia , Interações Microbianas , Mutação , Escherichia coli/genética , Nutrientes/fisiologiaRESUMO
BACKGROUND: Phosphate is a fundamental nutrient for all creatures. It is thus not surprising that a single bacterium carries different transport systems for this molecule, each usually operating under different environmental conditions. The phosphonate transport system of E. coli K-12 is cryptic due to an 8 bp insertion in the phnE ORF. RESULTS: Here we report that an E. coli K-12 strain carrying the triple knockout ΔpitA Δpst Δugp reverted the phnE mutation when plated on complex medium containing phosphate as the main phosphorus source. It is also shown that PhnCDE takes up orthophosphate with transport kinetics compatible with that of the canonical transport system PitA and that Pi-uptake via PhnCDE is sufficient to enable bacterial growth. Ugp, a glycerol phosphate transporter, is unable to take up phosphate. CONCLUSIONS: The phosphonate transport system, which is normally cryptic in E. coli laboratory strains is activated upon selection in rich medium and takes up orthophosphate in the absence of the two canonical phosphate-uptake systems. Based on these findings, the PhnCDE system can be considered a genuine phosphate transport system.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Fosfato/genética , Fosfatos/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , MutaçãoRESUMO
Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Ornitina/análogos & derivados , Fosfatos/deficiência , Vibrio cholerae/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Lipídeos , Lipídeos de Membrana/química , Mutação , Ornitina/metabolismo , Fosfatos/metabolismo , Vibrio cholerae/genéticaRESUMO
One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.
Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Periplásmicas de Ligação/genética , Proteínas de Ligação a Fosfato/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Vibrio cholerae O1/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Códon/química , Códon/metabolismo , Biologia Computacional , Endorribonucleases/genética , Endorribonucleases/metabolismo , Sequências Repetidas Invertidas , Óperon , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vibrio cholerae O1/metabolismo , Vibrio cholerae O1/patogenicidade , VirulênciaRESUMO
Phosphate homeostasis is tightly regulated in bacteria. Phosphate scarcity is overcome by inducing the expression of genes associated with the scavenging of phosphate and phosphate-containing molecules, while phosphate surplus is stored in the form of polyphosphate (polyP). Regulation of the genes involved in polyP metabolism was investigated. Knockout of the most distal gene of the pstSCAB-phoU operon that encodes a Pi-transport system results in large accumulation of polyphosphate (polyP). Here, we show that the phoU mutation differentially affects the transcription of ppk and ppx, that respectively, encode a polyP kinase and a polyP exopolyphosphatase, by increasing the former and reducing the latter, further contributing the accumulation of polyP. We also show that ppk forms an operon with the upstream gene hemB and that neither ppk nor ppx positively respond to Pi starvation. Furthermore, a putative PHO-box sequence in ppx regulatory region did not show a strong affinity for the PHO response regulator PhoB, while the promoter of hemB does not carry a PHO-box sequence. Altogether, the data indicate that the main genes involved in polyP metabolism, ppk and ppx, are differentially regulated in the absence of phoU, but neither gene belongs to the PHO regulon.
Assuntos
Regulação Bacteriana da Expressão Gênica , Fosfoproteínas Fosfatases/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Guanosina Tetrafosfato/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/metabolismo , Transcrição GênicaRESUMO
Chromobacterium violaceum is a free-living bacterium that inhabits low-nutrient environments such as the Amazon basin. Bacteria respond to phosphate (Pi) shortage by expressing a range of genes involved in Pi uptake and assimilation, known as the PHO regulon. Several PHO regulon genes have been annotated in the genome of C. violaceum. Here we show that C. violaceum is extremely well adapted to low-Pi conditions. Remarkably, this bacterium is able to grow in media containing only traces of Pi. The PHO regulon genes are induced upon Pi depletion, but the bacteria continued to grow under these conditions. Unlike other Proteobacteria hitherto analyzed, neither PstS nor PhoU play a role in the repression of the PHO regulon under Pi excess.