Evaluation of the cell growth of mycobacteria using Mycobacterium smegmatis mc2 155 as a representative species

The study of the in vitro cell growth of mycobacteria still remains a fastidious, difficult, and time-consuming procedure. In addition, assessing mycobacterial growth in the laboratory is often complicated by cell aggregation and slow growth-rate. We now report that the use of a stainless steel spring in the culture led to an absence of large cell clumps, to a decrease of dead cells in the exponential phase and to growth of a more homogeneous population of large cells. We also report that flow cytometry is a rapid, simple and reliable approach to monitor mycobacterial cell growth and viability. Here, we monitored Mycobacterium smegmatis cellular growth by optical density, dry cell mass, and colony forming units; in addition, viability, cell size and granularity profiles were analyzed by flow cytometry, and cell morphology by electron microscopy. Cultures monitored by flow cytometry may lead to a better understanding of the physiology of mycobacteria. Moreover, this methodology may aid in characterizing the cell growth of other fastidious species of microorganisms.     

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.     

Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Str?m, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.     

Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.     

Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.

The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.     

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.     

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.