Prototrophic Bacteria Research Articles

Coenzyme biosynthesis in response to precursor availability reveals incorporation of β-alanine from pantothenate in prototrophic bacteria

Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon sources and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and β-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for β-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

Vitamin B12 is not shared by all marine prototrophic bacteria with their environment

Vitamin B12 (cobalamin, herein B12) is an essential cofactor involved in amino acid synthesis and carbon resupply to the TCA cycle for most prokaryotes, eukaryotic microorganisms, and animals. Despite being required by most, B12 is produced by only a minor fraction of prokaryotes and therefore leads to complex interaction between prototrophs and auxotrophs. However, it is unknown how B12 is provided by prototrophs to auxotrophs. In this study, 33 B12 prototrophic alphaproteobacterial strains were grown in co-culture with Thalassiosira pseudonana, a B12 auxotrophic diatom, to determine the bacterial ability to support the growth of the diatom by sharing B12. Among these strains, 18 were identified to share B12 with the diatom, while nine were identified to retain B12 and not support growth of the diatom. The other bacteria either shared B12 with the diatom only with the addition of substrate or inhibited the growth of the diatom. Extracellular B12 measurements of B12-provider and B12-retainer strains confirmed that the cofactor could only be detected in the environment of the tested B12-provider strains. Intracellular B12 was measured by LC-MS and showed that the concentrations of the different B12-provider as well as B12-retainer strains differed substantially. Although B12 is essential for the vast majority of microorganisms, mechanisms that export this essential cofactor are still unknown. Our results suggest that a large proportion of bacteria that can synthesise B12de novo cannot share the cofactor with their environment.

Microbiological status of natural and anthropogenic soils of the Taganrog Bay coast at different levels of combined pollution with heavy metals and PAHs.

The effect of heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) pollution on the microbiological status of soils on the coast of the Taganrog Bay and adjacent areas was studied. The content of total and exchangeable forms of HMs, the content of 16 priority PAHs and the abundance of several groups of culturable microorganisms was determined, namely copiotrophic, prototrophic, aerobic spore-forming bacteria, actinomycetes, molds and yeasts. The content of total and exchangeable forms of HMs in urban coastal soils in industrial zone significantly exceeded that in non-urban soils. The maximum concentrations of total forms of Mn, Cr, Ni, Cu, Zn, Pb and Cd are 1821, 871, 143, 89, 1390, 317 and 10mg/kg, respectively. The median value of the total content of 16 PAHs in urban soils is 3 times higher than in the soils of natural areas and reached 4309ng/g. The lowest numbers of copiotrophic bacteria, prototrophic bacteria and aerobic spore-forming bacteria were found in the soils of industrial zone: 6.8, 13.8 and 0.63million CFUg-1 dry soil, respectively. The largest numbers of copiotrophic bacteria, prototrophic bacteria and aerobic spore-forming bacteria were recorded in the soils of natural areas-72.5, 136 and 5.73million CFUg-1 dry soil, respectively. It was found that the abundance of copiotrophs, prototrophs, and aerobic spore-forming bacteria is more affected by the urbanization of coastal soils including the pollution of HMs and PAHs. Other groups of microorganisms (actinomycetes, molds and yeasts) turned out to be more resistant to anthropogenic factors.

Vitamin Biosynthesis by Human Gut Butyrate-Producing Bacteria and Cross-Feeding in Synthetic Microbial Communities.

We investigated the requirement of 15 human butyrate-producing gut bacterial strains for eight B vitamins and the proteinogenic amino acids by a combination of genome sequence analysis and in vitro growth experiments. The Ruminococcaceae species Faecalibacterium prausnitzii and Subdoligranulum variabile were auxotrophic for most of the vitamins and the amino acid tryptophan. Within the Lachnospiraceae, most species were prototrophic for all amino acids and several vitamins, but biotin auxotrophy was widespread. In addition, most of the strains belonging to Eubacterium rectale and Roseburia spp., but few of the other Lachnospiraceae strains, were auxotrophic for thiamine and folate. Synthetic coculture experiments of five thiamine or folate auxotrophic strains with different prototrophic bacteria in the absence and presence of different vitamin concentrations were carried out. This demonstrated that cross-feeding between bacteria does take place and revealed differences in cross-feeding efficiency between prototrophic strains. Vitamin-independent growth stimulation in coculture compared to monococulture was also observed, in particular for F. prausnitzii A2-165, suggesting that it benefits from the provision of other growth factors from community members. The presence of multiple vitamin auxotrophies in the most abundant butyrate-producing Firmicutes species found in the healthy human colon indicates that these bacteria depend upon vitamins supplied from the diet or via cross-feeding from other members of the microbial community.IMPORTANCE Microbes in the intestinal tract have a strong influence on human health. Their fermentation of dietary nondigestible carbohydrates leads to the formation of health-promoting short-chain fatty acids, including butyrate, which is the main fuel for the colonic wall and has anticarcinogenic and anti-inflammatory properties. A good understanding of the growth requirements of butyrate-producing bacteria is important for the development of efficient strategies to promote these microbes in the gut, especially in cases where their abundance is altered. The demonstration of the inability of several dominant butyrate producers to grow in the absence of certain vitamins confirms the results of previous in silico analyses. Furthermore, establishing that strains prototrophic for thiamine or folate (butyrate producers and non-butyrate producers) were able to stimulate growth and affect the composition of auxotrophic synthetic communities suggests that the provision of prototrophic bacteria that are efficient cross feeders may stimulate butyrate-producing bacteria under certain in vivo conditions.

Generation of Enterobacter sp. YSU auxotrophs using transposon mutagenesis.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

An Engineered Methionyl‐tRNA Synthetase Enables Azidonorleucine Incorporation in Methionine Prototrophic Bacteria

Have you Met this? Methionine prototrophic E. coli harboring a single genomic copy of an engineered methionyl-tRNA synthetase (MetRS) gene, metG*, are more efficient at incorporating azidonorleucine (ANL) into recombinant proteins than prototrophic E. coli containing a multicopy plasmid expressing metG* and a genomic copy of the native MetRS (metG).

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe, Tyr and Trp.

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C(alpha/beta) positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D2O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62-79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.

New Thioredoxins and Glutaredoxins as Electron Donors of 3′-Phosphoadenylylsulfate Reductase

Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.

Correlation between structural diversity and catabolic versatility of metal-affected prototrophic bacteria in soil

Application of sewage sludge to an agricultural field resulted in contamination of Zn between 92 and 345 mg kg −1 of soil and other metals. The corresponding value of the control soil was 48 mg Zn kg −1. Metal effects on the structural diversity and the catabolic versatility of bacteria capable of growing in the absence of growth factors (prototrophs) were studied 6 yr after sludge application was discontinued. The number of strain clusters distinguished by amplified ribosomal DNA restriction analysis (ARDRA) was reduced by 39% when comparing isolates from the control and the most contaminated soil. Concomittantly, the average number of aromatic acids utilised per isolate from among 21 substrates tested decreased from 12.28 to 5.23. This loss in catabolic versatility was greater in Gram-negative (68%) than in Gram-positive bacteria (49%). Catabolic versatility, due to increasing metal stress, also decreased within ARDRA-clusters from different soils. As a consequence of metal stress, the proportion of frequent catabolic capabilities, partly related to energetically good aromatic C sources, increased, while the reverse was true for rare capabilities. Due to bioenergetic reasons discussed, it is supposed that the catabolically less versatile strains are more tolerant to metals than are the more versatile ones. The difference in the catabolic versatility between Gram-negative and Gram-positive bacteria and the greater loss of this property in the former may explain why, in metal contaminated soils, Gram-negatives are selected at the expense of Gram-positive bacteria.

Evidence that inhibitor(s) are formed which may interfere with the growth of revertant colonies in the ames salmonella and the E. Coli tryptophan reverse mutation assays when strictly anaerobic conditions are used

Spontaneous and chemically induced revertant colonies were not observed on plates when a strictly anaerobic environment and anaerobically prepared media were used to perform the Ames histidine reversion assay with each of eight different Salmonella strains. A similar effect was observed when the E. coli tryptophan reverse mutation assay was performed under strictly anaerobic conditions. We provide evidence here that under anaerobic conditions growth inhibitor(s) are formed by the S. typhimurium and E. coli bacteria when the limited histidine and tryptophan, respectively, are depleted from the medium. The inhibitor(s) are nonspecific and inhibit the growth not only of prototrophic bacteria but also of the inhibitor-producing bacteria as measured by neutralized supernatants of growth-limiting minimal liquid cultures. Inhibitor(s) are also formed in stationary phase cultures of Salmonella and E. coli in minimal liquid medium supplemented with excess histidine and tryptophan, respectively. These results suggest that inhibitor formation under anaerobic conditions is a physiological phenomenon which interferes with at least two reverse mutation assays. Whether or not it also interferes with the reverse mutagenesis process remains to be determined.

Assimilation efficiency and energy contents of prototrophic bacteria

AbstractWhen the assimilation efficiency in batch culture of a variety of heterotrophic bacteria was tested in media containing progressively smaller concentrations of carbon substrate, two groups emerged. Those in the first group assimilated carbon progressively more efficiently as the initial concentration in the culture medium was lowered below 2 mg C/ml; some displaying efficiencies exceeding 80%. Those in the second group assimilated carbon at rates that changed little, if at all, with changes in the initial concentrations of carbon substrate. Members of the second group differed from the first in their metabolic versatility as evidenced by the ability of each to catabolize hydrocarbons. The energy content (heat of combustion) of the cells of the species tested varied little with changes in cultured conditions, and values for all fell close to the average for bacteria (5411 cal/g ash‐free dry wt). In continuous aerobic culture on glucose‐minimal medium, the carbon assimilation and energy assimilation efficiencies were simultaneously maximal for Enterobacter aerogenes when the input carbon concentration was 0.2004 mg/ml and the dilution rate was 0.57. An equation for predicting heat of combustion of cells from knowledge of their C,H,N, and O ratios was devised and tested. Precision ranged from +6% to −16%.

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).

Plasmid Content and Tumor Initiation Complementation by Agrobacterium tumefaciens IIBNV6

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.

Carbon assimilation from simple and complex media by prototrophic heterotrophic bacteria

Carbon assimilation from simple and complex media by prototrophic heterotrophic bacteria

Thymineless mutants and their resistance to trimethoprim.

Trimethoprim was found to promote the uptake of thymine from Wellcotest Agar by prototrophic bacteria and by thymineless auxotrophs. However, the concentration of trimethoprim required was substantially higher in the case of the latter mutants. It was found that the minimum inhibitory concentration (MIC) of trimethoprim for thymineless mutants was very much higher than that obtained for thymine-independent bacteria providing that thymine or thymidine was present. From these results it is concluded that thymineless mutants are genuinely more resistant to trimethoprim. If levels of thymine derivatives sufficient for the growth of thymineless mutants are present in vivo then such mutants may be of therapeutic significance.

Ensaio sôbre métodos de obtenção de mutantes Auxotróficos em xanthomonas campestris

Different methods were tried with the aim of obtaining auxotrophic mutants from the phytopathogenic bacteria Xanthomonas campestris, isolated from Brassica oleraeea. The most effective method was by combining the method of Gorini and Kaufman, (using 0,54 M sucrose to avoid the lysis of prototrophic bacteria) and the method of Lubin (by recycling the growing of the bacterial cells with penicillin. Three types of mutants were produced: a) a group of unstable mutants; b) a group of mutants with residual growth; c) a group of auxotrophic mutants requiring one of the following aminoacids: proline, tryptophane, and phenylalanine; one mutant requiring isoleucine plus valine; one mutant requiring histidine and leucine; and one mutant requiring glycine and lysine. The mutant deficient for the synthesis of glycine and lysine showed a relationship toward ornithine and citrulline, since lysine could be substituted by either one of the latter aminoacids.

Incorporation of thymine into prototrophic and thymine-dependent mutants of Bacillus anthracis.

SUMMARY: Mutants of Bacillus anthracis which were prototrophic in a defined medium below 32° required either thymidine (mutant thy) or low concentration of thymine (mutant thytlr) for growth at 37°. No thymidylate synthetase was produced by these mutants at 37°. Normal enzyme activity was found in bacteria grown at 25°. Absence of thymidylate synthetase activity in bacteria grown at 37° could not be accounted for either by a heat-sensitive enzyme protein or by an inhibitor of this enzyme activity. The incorporation of thymine by prototrophic bacteria of Bacillus anthracis was negligible at either temperature. Mutant thytlr and its prototrophic revertant both readily took up thymine at 37° but not at 25°. Thymine uptake by both prototrophic and thy bacteria was markedly enhanced by the presence of any of the deoxyribonucleosides except thymidine. Moreover, thymidine antagonized thymine uptake. No thymidine phosphorylase activity was detected in B. anthracis, but phosphorolytic decomposition of deoxyuridine was found and that of deoxyadenosine seemed likely. The phosphorolytic reaction of deoxyuridine resulted in an accumulation of deoxyribose by thytlr bacteria, whilst both wild-type and thy bacteria catabolized this sugar. The secondary mutation in thy bacteria presumably resulted in a defect in synthesis of 1,5-phosphodeoxyribo mutase or deoxyribose 5-phosphate aldolase (E.C. 4.1.2.4). Although no thymidine phosphorylase was detected in B. anthracis, resting suspensions of this bacterium transferred the deoxyribosyl moiety from thymidine to thymine. This indicates the presence of the enzyme nucleoside purine (pyrimidine) deoxyribosyl transferase in B. anthracis. This particular enzyme may also be involved in the increased thymine uptake in the presence of different deoxyribonucleosides.