Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

Partial characterization of the cell walls ofMycobacterium leprae

Cell walls were prepared fromMycobacterium leprae (separated and purified from experimentally infected armadillo),M. tuberculosis, M. smegmatis, andMicrococcus lysodeikticus. The purity of the above wall preparations was confirmed after negative staining and shadow-casting and subsequent observation under the electron microscope. As judged from the electron microscopic observations, the bacteria were of different fragility in the following increasing order:M. tuberculosis, M. smegmatis, M. leprae, andMicro. lysodeikticus. The cell walls were hydrolyzed with 6N HCl, and the amino acids were identified by thin-layer chromatography compared with the authentic standards. With the same purification procedures, it was not possible to obtain satisfactorily pure peptidoglycan fromM. leprae. In leprosy bacilli,meso-DAP was found to be present, ín the walls; however, contamination by nonwall amino acids did not allow the confirmation of previous results, a finding that suggests that glycine completely replaced L-alanine inM. leprae cell walls.     

Suitability of lipid materials for culture ofMalassezia as evaluated from its cellular fatty acid composition

Malassezia is a facultative or obligatory lipophilic yeast. We devised new lipid-supplemented media suitable for the culture ofMalassezia. Malassezia furfur andM. pachydermatis grew well on both solid and liquid media supplemented with creaming powder preparations which are commercially available at moderate prices. Striking differences were found between the cellular fatty acid compositions ofM. furfur grown on media supplemented with creaming powder and that grown on media with conventional olive oil.Malassezia furfur grown on media with olive oil had nearly the same fatty acid composition as olive oil, with C18:1 amounting to 80%, while that grown on media supplemented with creaming powder had C16, C18:1 and C18:2 as the principal components. The use of these supplementary lipids appeared not to inhibit the normal synthesis of fatty acid inM. furfur. For the culture ofM. pachydermatis, media supplemented with creaming powder were also found more suitable than lipid-free media. The media devised are considered excellent, because they appear to provide a more natural growth environment forMalassezia.     

An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins

Summary We have analyzed and compared the amino acid sequences of the type 4 fimbrial subunits fromPseudomonas aeruginosa, Moraxella bovis, M. nonliquefaciens, Bacteroides nodosus, Neisseria gonorrhoeae, andN. meningitidis. We propose a consensus sequence for the highly conserved aminoterminal regions of these proteins. In the variable regions, a domain corresponding to an epitope common toN. gonorrhoeae andN. meningitidis fimbriae is conserved, both in sequence and in environment, in fimbrial subunits fromB. nodosus. The subunits fromM. bovis andP. aeruginosa do not show any homologies to this sequence. In all of the subunits, the carboxy-terminal half of the molecule consists of a series of fairly hydrophobic domains. The last three domains, two of which include the cysteines of the disulfide bridge inN. gonorrhoeae, P. aeruginosa, andM. bovis, are more or less conserved in sequence in all of the proteins including that ofB. nodosus. We propose that these conserved hydrophobic regions, which have the potential to form a series of beta-sheets, form a structural framework around which more variable hydrophilic sequences determining immunological profile are arranged. The evolutionary relationships of the contemporary proteins and the distribution of type 4 fimbriae are also discussed.     

Effect of 2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone onMycobacterium smegmatis

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells ofMycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associate d with this cycle (glutamate, asparagine). DDA inhibits the incorporation of both¹⁴C-leucine and¹⁴C-adenine into appropriate macromolecules ofM. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity ofU-¹⁴C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth ofM. smegmatis probably through an interference with the cell energy-carbon metabolism.     

Dynamics of growth of atypical mycobacteria in different liquid cultivation media

The dynamics of growth ofMycobacterium smegmatis, M. fortuitum andM. phlei in liquid media used also for cultivation of typical mycobacteria (Sauton, Youmans, Kirchner, Šula) was compared with that in Davis and Merrill media. In the Merrill medium glucose (as the only organic component) was replaced with another carbon source and the effect of this modification was investigated. The results obtained show that the Merrill medium, its modification in particular, is suitable for cultivation ofM. smegmatis andM. fortuitum. 2-Oxoglutarate and succinate are important as the sole carbon sources in the case ofM. fortuitum andM. phlei respectively.     

Construction and use of integrative vectors to express foreign genes in mycobacteria

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained.     

Absolute Stereochemistry of Analogs of Optical Active α-Ionylideneacetic Acids

Two kinds of iron-containing proteins the molecular masses of which were about 10 kDa and 24 kDa were isolated from cytoplasmic fractions of Mycobacterium smegmatis grown under iron-sufficient (50 μM Fe) and iron-overload (500 μM Fe) conditions. Based upon the elution profiles in two chromatographic systems, spectrophotometric analysis, and ESR spectrum measurement, the protein of 10 kDa met the criteria for classification as a ferredoxin. Another protein of 24 kDa showed no enzymatic activity, though its detailed structure was unknown. The ferredoxin and the protein of 24 kDa contained about 30% and 50% of the total cellular iron, respectively, when cells were grown under the above conditions. The synthesis of the protein of 24 kDa was, however, completely repressed in cells grown under iron-deficient (0.5 μM Fe) conditions, although the ferredoxin was still synthesized to some extent even in iron-deficient cells. These results suggested that both ferredoxin and the protein of 24 kDa could be synergistically involved in iron storage in this organism.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Extraction and preliminary characterizaion of a mycobacteriolytic preparation (stazyme) from aStaphylococcus strain: mycobactericidal activity and its use in rapid extraction of mycobacterial DNA

The clear culture filtrate from 1-L culture of a laboratory contaminant ofStaphylococcus (coagulase^– strain, designated Clavelis) was filtered, concentrated, dialyzed, and the proteins were precipitated. The precipitate was washed, concentrated, and aliquoted (about 4 mg of total proteins/ml, designated as Stazyme). The crude preparation was subjected to gel filtration on Sephadex G-75, and the fractions were screened for their lytic ability againstMycobacterium smegmatis. Native proteins in the stazyme were electrophoretically separated, electroeluted, and their lytic activity againstM. smegmatis was compared with parallel controls (partially purified proteins extracted from the same quantity of the uninoculated bacterial growth medium). Only stazyme preparations caused significant growth inhibition ofM. smegmatis, M. chelonae, M. xenopi, M. tuberculosis, andM. kansasii. Stazyme essentially possessed a lytic activity measured with purifiedM. smegmatis andMicrococcus lysodeikticus cell walls and showed high bactericidal activity againstM. smegmatis, M. chelonae, andM. tuberculosis. It was also able to rapidly lyse intactM. smegmatis organisms, permitting significant yield of mycobacterial DNA.     

Effect of isoniazid on glutathione biosynthesis and degradation inMycobacterium smegmatis

The mechanism of glutathione (GSH) depletion by isoniazid (INH) was studied inM. smegmatis. INH increased the activity of γ-glutamyl transferase (GGT) whether added before medium inoculation or to actively growing cells. The activity of GGT in cells grown from the beginning in INH-containing medium increased significantly on growth days 2–6. Three-day oldM. smegmatis cells treated with INH exhibited a 30–65 % increase in the activity of GGT. The activities of γ-glutamyl-cysteine synthase (GGCS) and GSH synthase (GS) were lowered by 50 and 56 % respectively on the second day of growth whenM. smegmatis was grown in a medium supplemented with 1.5 mg INH per L. In 3-day oldM. smegmatis, INH significantly inhibited the activities of GSH biosynthetic enzymes. The results demonstrate that the increased activity of GGT and decreased activities of GSH biosynthetic enzymes are responsible for GSH depletion by INH inM. smegmatis.     

The chromosomal distribution of the major and minor satellite is not conserved in the genusMus

The cytological distribution of the major and minor satellite first identified inMus musculus was studied in the karyotypes of three related subspecies and two other species of the genusMus. Both the major and minor satellite showed species dependent hybridization patterns. The major satellite is confined to the centromere region inM. musculus and related subspecies. However, inM spretus andM. caroli, the chromosomal arm regions contain this sequence class. In contrast the minor satellite is found at the kinetochore region inM. musculus and related subspecies but is distributed throughout the entire centromeric domain inM. spretus and appears to be excluded from the chromosomes ofM. caroli. There is an apparent correlation between the chromosomal location of these satellites and their phylogenetic relationship. Determination of the biological roles of the major and minor satellites fromM. musculus must take into account their differential chromosomal distribution in otherMus species.     

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

Summary The existence of Ca²⁺-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.     

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house miceMus domesticus andMus musculus

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation inMus domesticus and inMus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed withM. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230–240 bp repetitive unit of theM. domesticus stDNA. In contrast, EcoRI digestion reveals thatM. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by anM. domesticus stDNA probe) shows different patterns in theM. domesticus andM. musculus karyotypes, with about 60% ofM. domesticus stDNA retained in theM. musculus genome. It is particularly noteworthy that the pericentromeric regions ofM. musculus chromosomes 1 and X are totally devoid ofM. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in theM. domesticus stDNA, as if they are organized in tandem repeats longer than those ofM. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230–240 bp stDNA repetitive unit through the amplification, in theM. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: Evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages

The intracellular growth kinetics ofMycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains ofMycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMMØ) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMMØ. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMMØ than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth ofM. tuberculosis H37Rv andM. avium was compared inside BMMØ from both theBcg ^s (C57BL/6) andBcg ^r (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained withM. tuberculosis-orM. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMMØ to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.     

Translation enhancement by optimized downstream box sequences in <Emphasis Type="Italic">Escherichia coli </Emphasis>and<Emphasis Type="Italic"> Mycobacterium smegmatis</Emphasis>

The effect on translation of downstream box sequences optimized for binding to Mycobacterium smegmatis and Escherichia coli 16S rRNA in the absence of a Shine--Dalgarno (SD) region was investigated. The relative translational efficiency of each construct in either M. smegmatis or E. coli was determined. Eradication of the SD region in the absence of a downstream box abolished the translation activity. In contrast, optimized downstream box constructs resulted in a 13- and 18-fold increase in protein synthesis, relative to non-optimized DB controls in E. coli and M. smegmatis, respectively.     

The monomeric and dimeric mannose-binding proteins from the Orchidaceae speciesListera ovata andEpipactis helleborine: sequence homologies and differences in biological activities

The Orchidaceae speciesListera ovata andEpipactis helleborine contain two types of mannose-binding proteins. Using a combination of affinity chromatography on mannose-Sepharose-4B and ion exchange chromatography on a Mono-S column eight different mannose-binding proteins were isolated from the leaves ofListera ovata. Whereas seven of these mannose-binding proteins have agglutination activity and occur as dimers composed of lectin subunits of 11–13 kDa, the eighth mannose-binding protein is a monomer of 14 kDa devoid of agglutination activity. Moreover, the monomeric mannose-binding protein does not react with an antiserum raised against the dimeric lectin and, in contrast to the lectins, is completely inactive when tested for antiretroviral activity against human immunodeficiency virus type 1 and type 2. Mannose-binding proteins with similar properties were also found in the leaves ofEpipactis helleborine. However, in contrast toListera only one lectin was found inEpipactis. Despite the obvious differences in molecular structure and biological activities molecular cloning of different mannose-binding proteins fromListera andEpipactis has shown that these proteins are related and some parts of the sequences show a high degree of sequence homology indicating that they have been conserved through evolution.Abbreviations EHMBP Epipactis helleborine mannose-binding protein - LOMBP Listera ovata mannose-binding protein Note: The nucleotide sequences reported in this paper will appear in the Genbank/EMBL Data library with the accession numbers L18894, L18895 and U07787.