scholarly journals Mycobacterium smegmatisl-Alanine Dehydrogenase (Ald) Is Required for Proficient Utilization of Alanine as a Sole Nitrogen Source and Sustained Anaerobic Growth

2002 ◽  
Vol 184 (18) ◽  
pp. 5001-5010 ◽  
Author(s):  
Zhengyu Feng ◽  
Nancy E. Cáceres ◽  
Gautam Sarath ◽  
Raúl G. Barletta
Keyword(s):  
Enzyme Activity ◽  
Nitrogen Source ◽  
Type Strain ◽  
Growth Stage ◽  
Reductive Amination ◽  
Wild Type Strain ◽  
Anaerobic Growth ◽  
Sole Nitrogen Source ◽  
Wild Type ◽  
Anaerobic Conditions

ABSTRACT NAD(H)-dependent l-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of l-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of l- or d-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with l-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.

scholarly journals The effect of thiourea on ureide metabolism in Neurospora crassa

1973 ◽  
Vol 136 (3) ◽  
pp. 749-755 ◽  
Author(s):  
Jasti Nirmala ◽  
Killampalli Sivarama Sastry
Keyword(s):  
Neurospora Crassa ◽  
Nitrogen Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Inorganic Nitrogen ◽  
Wild Strain ◽  
Sole Nitrogen Source ◽  
Wild Type ◽  
Selective Toxicity ◽  
In The Wild

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.

scholarly journals A Mutation in the 3-Phosphoglycerate Kinase Gene Allows Anaerobic Growth of Bacillus subtilis in the Absence of ResE Kinase

1999 ◽  
Vol 181 (22) ◽  
pp. 7087-7097 ◽  
Author(s):  
Michiko M. Nakano ◽  
Yi Zhu ◽  
Koki Haga ◽  
Hirofumi Yoshikawa ◽  
Abraham L. Sonenshein ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Anaerobic Respiration ◽  
Phosphoglycerate Kinase ◽  
Anaerobic Growth ◽  
Wild Type ◽  
Kinase Gene ◽  
Glycolytic Intermediate

ABSTRACT The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resDmutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1,3-diphosphoglycerate, is responsible for the observed effect of thepgk-1 mutation on anaerobiosis of resE mutant cells.

scholarly journals Induction and repair of DNA strand breaks in Bacteroides fragilis

1990 ◽  
Vol 36 (7) ◽  
pp. 490-494 ◽  
Author(s):  
Valerie R. Abratt ◽  
Meyrick J. Peak ◽  
Jennifer G. Peak ◽  
Joseph D. Santangelo ◽  
David R. Woods
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Bacteroides Fragilis ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Sensitive Mutant ◽  
Strand Breaks ◽  
In The Wild ◽  
Strand Breakage ◽  
Dna Strand

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair. Key words: Bacteroides fragilis, DNA breakage, DNA repair, caffeine.

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein

1978 ◽  
Vol 24 (5) ◽  
pp. 629-631 ◽  
Author(s):  
La Verne Russell ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Wild Type Strain ◽  
Anaerobic Growth ◽  
Receptor Protein ◽  
Wild Type ◽  
Cyclic Amp Receptor Protein ◽  
E Coli ◽  
Cyclic Amp Synthesis

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

scholarly journals Transcriptional and Proteomic Analysis of a Ferric Uptake Regulator (Fur) Mutant of Shewanella oneidensis: Possible Involvement of Fur in Energy Metabolism, Transcriptional Regulation, and Oxidative Stress

2002 ◽  
Vol 68 (2) ◽  
pp. 881-892 ◽  
Author(s):  
Dorothea K. Thompson ◽  
Alexander S. Beliaev ◽  
Carol S. Giometti ◽  
Sandra L. Tollaksen ◽  
Tripti Khare ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcriptional Regulation ◽  
Energy Metabolism ◽  
Type Strain ◽  
Wild Type Strain ◽  
Shewanella Oneidensis ◽  
Anaerobic Growth ◽  
Ferric Uptake Regulator ◽  
Wild Type ◽  
And Oxidative Stress

ABSTRACT The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.

scholarly journals A Conserved Histidine in Cytochrome c Maturation Permease CcmB of Shewanella putrefaciens Is Required for Anaerobic Growth below a Threshold Standard Redox Potential

2006 ◽  
Vol 189 (3) ◽  
pp. 1036-1043 ◽  
Author(s):  
Jason R. Dale ◽  
Roy Wade ◽  
Thomas J. DiChristina
Keyword(s):  
Electron Acceptor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Shewanella Putrefaciens ◽  
Electron Acceptors ◽  
Anaerobic Growth ◽  
Histidine Residue ◽  
Wild Type ◽  
Standard Redox Potential ◽  
Wide Range

ABSTRACT Shewanella putrefaciens strain 200 respires a wide range of compounds as terminal electron acceptor. The respiratory versatility of Shewanella is attributed in part to a set of c-type cytochromes with widely varying midpoint redox potentials (E′0). A point mutant of S. putrefaciens, originally designated Urr14 and here renamed CCMB1, was found to grow at wild-type rates on electron acceptors with high E′0 [O2, NO3 −, Fe(III) citrate, MnO2, and Mn(III) pyrophosphate] yet was severely impaired for growth on electron acceptors with low E′0 [NO2 −, U(VI), dimethyl sulfoxide, TMAO (trimethylamine N-oxide), fumarate, γ-FeOOH, SO3 2−, and S2O3 2−]. Genetic complementation and nucleotide sequence analyses indicated that the CCMB1 respiratory mutant phenotype was due to mutation of a conserved histidine residue (H108Y) in a protein that displayed high homology to Escherichia coli CcmB, the permease subunit of an ABC transporter involved in cytochrome c maturation. Although CCMB1 retained the ability to grow on electron acceptors with high E′0, the cytochrome content of CCMB1 was <10% of that of the wild-type strain. Periplasmic extracts of CCMB1 contained slightly greater concentrations of the thiol functional group (-SH) than did the wild-type strain, an indication that the Eh of the CCMB1 periplasm was abnormally low. A ccmB deletion mutant was unable to respire anaerobically on any electron acceptor, yet retained aerobic respiratory capability. These results suggest that the mutation of a conserved histidine residue (H108) in CCMB1 alters the redox homeostasis of the periplasm during anaerobic growth on electron acceptors with low (but not high) E′0. This is the first report of the effects of Ccm deficiencies on bacterial respiration of electron acceptors whose E′0 nearly span the entire redox continuum.

scholarly journals Expression and Regulation of a Silent Operon, hyf, Coding for Hydrogenase 4 Isoenzyme in Escherichia coli

2004 ◽  
Vol 186 (2) ◽  
pp. 580-587 ◽  
Author(s):  
William T. Self ◽  
Adnan Hasona ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Wild Type Strain ◽  
Receptor Protein ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Heterologous Promoter ◽  
Cyclic Amp Receptor Protein ◽  
Hydrogenase 3

ABSTRACT On the basis of hyf-lacZ fusion studies, the hyf operon of Escherichia coli, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the hyc-encoded hydrogenase 3 isoenzyme) activated hyf-lacZ. HyfR, an FhlA homolog encoded by the hyfR gene present at the end of the hyf operon, also activated transcription of hyf-lacZ but did so only when hyfR was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H2 production. Optimum expression of hyf-lacZ required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.

scholarly journals L-Phenylalanine Transport in Saccharomyces cerevisiae: Participation of GAP1, BAP2, and AGP1

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel A. Sáenz ◽  
Mónica S. Chianelli ◽  
Carlos A. Stella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ammonium Ion ◽  
Wild Type ◽  
Synthetic Media ◽  
Phenylalanine Transport

We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.

scholarly journals Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

2012 ◽  
Vol 79 (2) ◽  
pp. 678-687 ◽  
Author(s):  
Audrey Parangan-Smith ◽  
Steven Lindow
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Source ◽  
Pseudomonas Syringae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Nitrate Assimilation ◽  
Wild Type ◽  
Content Type ◽  
Nitrogenous Compounds

ABSTRACTThe ability ofPseudomonas syringaepv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant,P. syringaeΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasBmutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting thatP. syringaeexperiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-basedP. syringaenitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation ofP. syringaeconsumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.

scholarly journals Identification of Four Secreted Aspartic Protease-Like Proteins Associated With Sophorolipids Synthesis in Starmerella bombicola CGMCC 1576

2021 ◽  
Vol 12 ◽  
Author(s):  
Jun Liu ◽  
Guoqin Zhao ◽  
Xinyu Zhang ◽  
Xin Song
Keyword(s):  
Nitrogen Source ◽  
Gene Cluster ◽  
Yeast Extract ◽  
Type Strain ◽  
Wild Type Strain ◽  
Aspartic Protease ◽  
Wild Type ◽  
Pathogenic Yeast ◽  
Starmerella Bombicola ◽  
Key Genes

The non-pathogenic yeast Starmerella bombicola CGMCC 1576 is an efficient producer of sophorolipids (SLs). The lactonic SLs are mainly produced with yeast extract, and the acidic SLs are mainly produced with ammonium sulfate. Naturally produced SLs are a mixture of various lactonic and acidic SLs. Usually, the SL mixture is not well separated technically, and the separation cost is relatively high. In order to reduce the cost of separation, four secreted aspartic protease-like proteins were identified through proteomic analysis of fermentation broth of S. bombicola under different nitrogen source conditions. The coding genes of the four proteins, namely, sapl1, sapl2, sapl3, and sapl4, are of high sequence similarity (above 55%) and included in a gene cluster. The expression of the four genes was significantly upregulated on (NH4)2SO4 compared with that on yeast extract. The four genes were deleted together to generate a strain Δsapl. The titer of SLs in Δsapl reached 60.71 g/L after 5 days of fermentation using (NH4)2SO4 as the nitrogen source and increased by 90% compared with the wild-type strain. The concentration of acidic SLs was 55.84 g/L, accounting for 92% of the total SLs. The yield of SLs from glucose (g/g) by Δsapl was 0.78, much higher than that by wild-type strain (0.47). However, no increase of SLs production was observed in Δsapl under yeast extract condition. Compared with that of the wild-type strain, the expression levels of the key genes for SLs synthesis were all upregulated to varying degrees in Δsapl under (NH4)2SO4 conditions, and particularly, the expression level of ugta1 encoding UDP glucosyltransferase was upregulated by 14.3-fold. The results suggest that the sapl gene cluster is negatively involved in the production of SLs in the case of (NH4)2SO4 by restraining the expression of the key genes involved in SLs synthesis. The Δsapl strain is an excellent producer of high-titer and high-yield acidic SLs.

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