Gene putA Research Articles

NsiR3, a nitrogen stress-inducible small RNA, regulates proline oxidase expression in the cyanobacterium Nostoc sp. PCC 7120.

NsiR3 (nitrogen stress-inducible RNA 3) is a small noncoding RNA strongly conserved in heterocyst-forming cyanobacteria. In Nostoc sp. PCC 7120, transcription of NsiR3 is induced by nitrogen starvation and depends on the global nitrogen regulator NtcA. A conserved NtcA-binding site is centered around position -42.5 with respect to the transcription start site of NsiR3 homologs, and NtcA binds invitro to a DNA fragment containing this sequence. In the absence of combined nitrogen, NsiR3 expression is induced in all cells along the Nostoc filament but much more strongly in heterocysts, differentiated cells devoted to nitrogen fixation. Co-expression analysis of transcriptomic data obtained from microarrays hybridized with RNA obtained from Nostoc wild-type or mutant strains grown in the presence of ammonium or in the absence of combined nitrogen revealed that the expression profile of gene putA (proline oxidase) correlates negatively with that of NsiR3. Using a heterologous system in Escherichiacoli, we show that NsiR3 binds to the 5'-UTR of putA mRNA, resulting in reduced expression of a reporter gene. Overexpression of NsiR3 in Nostoc resulted in strong reduction of putA mRNA accumulation, further supporting the negative regulation of putA by NsiR3. The higher expression of NsiR3 in heterocysts versus vegetative cells of the N2 -fixing filament could contribute to the previously described absence of putA mRNA and of the catabolic pathway to produce glutamate from arginine via proline specifically in heterocysts. Post-transcriptional regulation by NsiR3 represents an indirect NtcA-operated regulatory mechanism of putA expression. DATABASE: Microarray data are available in GEO database under accession numbers GSE120377 and GSE150191.

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus

Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ΔputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.

Proline addition increases the efficiency of l‐lysine production by Corynebacterium glutamicum

The wild type of Corynebacterium glutamicum does not excrete l‐lysine, but mutants are developed to use C. glutamicum for the large‐scale production of this amino acid. l‐lysine is derived from oxaloacetate and pyruvate. A reduced entry of these precursors into the citric acid cycle triggers l‐lysine synthesis, but there is simultaneously an unfavorable reduction in growth rate. Here, we further investigated strains with reduced citrate synthase activity and observed that proline supplementation partly reversed the growth rate reduction, and simultaneously increased l‐lysine accumulation by 10%. The combined effect of faster growth and higher l‐lysine formation lead to an increase in l‐lysine productivity of about 14%. Small scale bioreactor cultivations showed an increase of the glucose uptake rate from 3.96 to 5.12 mmol gCDW−1 h−1 as the effect of proline addition. Deletion of the proline‐utilization gene putA demonstrated that proline did not serve to refill the citric acid cycle. The combined data show that proline addition has a favorable effect on l‐lysine formation and sugar uptake, which indicates that engineering sugar uptake is a future option for increased product formation.

Structural Basis of the Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon–helix–helix domain (PutA52, a polypeptide corresponding to residues 1–52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25-Å-resolution crystal structure of PutA52 bound to one of the operators (operator 2; 21 bp) shows that the protein contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic, with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and a 15-fold-lower affinity, showing that base pairs outside of the consensus motif impact binding. Structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nm, which is slightly higher than that of the PutA-DNA complex (K(d) approximately 45 nm). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nm for the PutA47-DNA complex. Thus, N-terminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a.

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.

Membrane association of proline dehydrogenase in Escherichia coli is redox dependent.

The PutA protein, product of the Escherichia coli gene putA, has two functions essential for proline utilization and for the regulation of putP and putA expression: as the peripheral membrane flavoprotein, proline dehydrogenase (EC 1.5.99.8), it transfers electrons from proline to the respiratory chain, and, as a repressor, it controls expression of genes putP and putA in response to proline supply. Association of proline dehydrogenase with the membrane was shown to require the simultaneous presence of the soluble enzyme, membranes, and proline. The kinetics of that association, monitored by following proline oxidation in a coupled enzyme assay system, were not altered when the transmembrane proton gradient generated during proline oxidation was dissipated by a proton ionophore. However, D-lactate or NADH could replace proline as a promoter of proline dehydrogenase-membrane association under anaerobic reaction conditions. These data imply that reduction of proline dehydrogenase and/or a membrane constituent promotes enzyme-membrane association. A biochemical mechanism is suggested whereby the concentration of proline dehydrogenase associated with the respiratory chain would be determined by proline supply.