Arginine and Citrulline Catabolic Pathways Encoded by the arc Gene Cluster of Lactobacillus brevis ATCC 367.

High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.

Characterization of the mycobacterial acyl-CoA carboxylase holo complexes reveals their functional expansion into amino acid catabolism.

Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-ß unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.

Physiology and substrate specificity of two closely related amino acid transporters, SerP1 and SerP2, of Lactococcus lactis.

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports L-serine, L-threonine, and L-cysteine with high affinity. Affinity constants (Km) are in the 20 to 40 µM range. SerP2 is a DL-alanine/DL-serine/glycine transporter. The preferred substrate appears to be DL-alanine for which the affinities were found to be 38 and 20 µM for the D and L isomers, respectively. The common substrate L-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 µM, respectively. Growth experiments demonstrate that SerP1 is the main L-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates L-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic D-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of D-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.

ArcD1 and ArcD2 Arginine/Ornithine Exchangers Encoded in the Arginine Deiminase Pathway Gene Cluster of Lactococcus lactis.

UNLABELLED: The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. Deletion of arcD1 resulted in loss of the growth advantage observed in the presence of high l-arginine in different growth media. Uptake of l-arginine and l-ornithine by resting cells was reduced to the low level observed for an ArcD1/ArcD2 double deletion mutant. Deletion of the arcD2 gene did not affect the growth enhancement, and uptake activities were slightly reduced. Nevertheless, recombinant expression of ArcD2 in the ArcD1/ArcD2 double mutant did recover the growth advantage. Kinetic characterization of ArcD1 and ArcD2 showed high affinities for both l-arginine and l-ornithine (Km in the micromolar range). A difference between the two transporters was the significantly lower affinity of ArcD2 for the cationic amino acids l-ornithine, l-lysine, and l-histidine. In contrast, the affinity of ArcD2 was higher for the neutral amino acid l-alanine. Moreover, ArcD2 efficiently translocated l-alanine, while ArcD1 did not. Both transporters revealed affinities in the mM range for agmatine, cadaverine, histamine, and putrescine. These amines bind but are not translocated. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway and that ArcD2 is not functionally expressed in the media used. ArcD2 is proposed to function together with the arcT gene that encodes a putative transaminase and is found adjacent to the arcD2 gene. IMPORTANCE: The arginine deiminase (ADI) pathway gene cluster in Lactococcus lactis contains two copies of a gene encoding an l-arginine/l-ornithine exchanger, the arcD1 and arcD2 genes. The physiological function of ArcD1 and ArcD2 was studied by deleting the two genes. It is concluded that ArcD1 is the main l-arginine/l-ornithine exchanger in the ADI pathway. ArcD2 is proposed to function as a l-arginine/l-alanine exchanger in a pathway together with the arcT gene, which is found adjacent to the arcD2 gene in the ADI gene cluster.

Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain.

BACKGROUND: The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1. RESULTS: In order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1ΔC. GroEL1ΔC, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1ΔC is no longer present in protein samples purified from the groEL1ΔC expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain. CONCLUSIONS: This novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.

Convergent evolution of the arginine deiminase pathway: the ArcD and ArcE arginine/ornithine exchangers.

The arginine deiminase (ADI) pathway converts L-arginine into L-ornithine and yields 1 mol of ATP per mol of L-arginine consumed. The L-arginine/L-ornithine exchanger in the pathway takes up L-arginine and excretes L-ornithine from the cytoplasm. Analysis of the genomes of 1281 bacterial species revealed the presence of 124 arc gene clusters encoding the pathway. About half of the clusters contained the gene encoding the well-studied L-arginine/L-ornithine exchanger ArcD, while the other half contained a gene, termed here arcE, encoding a membrane protein that is not a homolog of ArcD. The arcE gene product of Streptococcus pneumoniae was shown to take up L-arginine and L-ornithine with affinities of 0.6 and 1 µmol/L, respectively, and to catalyze metabolic energy-independent, electroneutral exchange. ArcE of S. pneumoniae could replace ArcD in the ADI pathway of Lactococcus lactis and provided the cells with a growth advantage. In contrast to ArcD, ArcE catalyzed translocation of the pathway intermediate L-citrulline with high efficiency. A short version of the ADI pathway is proposed for L-citrulline catabolism and the presence of the evolutionary unrelated arcD and arcE genes in different organisms is discussed in the context of the evolution of the ADI pathway.

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation.

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.

Cell division site placement and asymmetric growth in mycobacteria.

Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.

The chitobiose-binding protein, DasA, acts as a link between chitin utilization and morphogenesis in Streptomyces coelicolor.

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

Loss of the controlled localization of growth stage-specific cell-wall synthesis pleiotropically affects developmental gene expression in an ssgA mutant of Streptomyces coelicolor.

Members of the family of SsgA-like proteins (SALPs) are found exclusively in sporulating actinomycetes, and SsgA itself activates sporulation-specific cell division. We previously showed that SALPs play a chaperonin-like role in supporting the function of enzymes involved in peptidoglycan maintenance (PBPs and autolysins). Here we show that SsgA localizes dynamically during development, and most likely marks the sites where changes in local cell-wall morphogenesis are required, in particular septum formation and germination. In sporogenic aerial hyphae, SsgA initially localizes as strong foci to the growing tips, followed by distribution as closely spaced foci in a pattern similar to an early stage of FtsZ assembly. Spore septa formed in these hyphae colocalize with single SsgA-GFP foci, and when the maturing spores are separated, these foci are distributed symmetrically, resulting in two foci per mature spore. Evidence is provided that SsgA also controls the correct localization of germination sites. Transcriptome analysis revealed that expression of around 300 genes was significantly altered in mutants in ssgA and its regulatory gene ssgR. The list includes surprisingly many known developmental genes, most of which were upregulated, highlighting SsgA as a key player in the control of Streptomyces development.

The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development.

Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N-acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.

MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores.

MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

SsgA-like proteins determine the fate of peptidoglycan during sporulation of Streptomyces coelicolor.

During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process in actinomycetes, including that of the SsgA-like proteins (SALPs). Mutants for each of the individual SALP genes were obtained, and high-resolution and fluorescence imaging revealed that each plays an important and highly specific role in the control of the sporulation process, and their function relates to the build-up and degradation of septal and spore-wall peptidoglycan. While SsgA and SsgB are essential for sporulation-specific cell division in Streptomyces coelicolor, SsgC-G are responsible for correct DNA segregation/condensation (SsgC), spore wall synthesis (SsgD), autolytic spore separation (SsgE, SsgF) or exact septum localization (SsgG). Our experiments paint a picture of a novel protein family that acts through timing and localization of the activity of penicillin-binding proteins and autolysins, thus controlling important steps during the initiation and the completion of sporulation in actinomycetes.

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant.

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.