Abstract: Background and Objectives: Mycolyl-transferases are a clan of proteins that are especially present in the CMN genera (Corynebacterium, Mycobacterium and Nocardia), mycolyl-transferases are responsible for cell wall components synthesis. The mycobacteria and corynebacteria envelopes share some gross structural features and similar cell wall architecture. The aim of the present work is to identify C. glutamicum genes encoding for glycosyl-transferases enzymes activity. Materials and Methods: In silico search for “glycosyl transferases” that were common both to M. tuberculosis and Corynebacterium difteriae revealed the presence of PimA-like sequences in the actinomycete Streptomyces coelicolor and in the extremophile archeons Pyrococcus horikoshii, Aeropyrum pernix and Pyrococcus abyssi. Results: Highly conserved regions obtained permitted the design of mixtures of oligonucleotides pairs intended to PCR amplification of a pimA gene fragment. The integration of the internal pimA gene fragment at the bacterial genome was done by a single homologous recombination event at the identical wild-type pimA gene of C. glutamicum Or2262. The pimA gene (belonging to the locus pgsA-htrB-pimA) and encoding for glycosyl-transferase enzyme activity of the species C. glutamicum ATCC13032 and C. glutamicum sp. 2262 (reclassified as C. glutamicum Or2262) was successfully cloned. Conclusion: A comparison of the transformability of C. glutamicum Or2262 and f. C. glutamicum ATCC13032 RES167 revealed 18.0 times difference in the ratio of transformability, which suggested that it is attributed to the difference in the efficiency of plasmid-host recombination rather than the efficiency of diffusion of the plasmid through the bacterial envelope.
INTRODUCTION
Around 33% of the total populace is infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB) which remains the leading reason for mortality from a single infectious organism. The steadfastness of this pathogen is linked with its unmistakable cell divider structure. Mycobacterium tuberculosis has an odd lipid-rich cell wall including an ample collection of antigens, providing a protective hydrophobic leak-proof barrier against antibiotic drugs. This sophisticated and unusual structure is important for the growth, viability and infectious ability of this micro-organism thus, attracting attention as a goal for developing drugs and vaccines1,2.
A good deal of the available information of the functional features of the Corynebacterium envelope is derived from studies of the secretion of metabolites of economic importance by non-pathogenic strains from the Corynebacterium glutamicum group. Most of the evidence on the chemical and structural features, the Corynebacterum genus come from pathogenic strains studied in the context of the mycobacterial pathogenesis and from extrapolations from the important amount of knowledge on the mycobacterial envelope3-5. A more specific view of the corynebacterial envelope has been proposed in an experimental article that reviews the current status of corynebacterial structural research6.
Although, they are Gram-positive bacteria, corynebacteria and closely related micro-organisms share with enteric Gram-negative bacteria, the property having a strong permeability barrier other than the plasma membrane. In Gram-negative micro-organisms this additional barrier is accounted for by a complex outer layer of structure and composition different to other bacterial plasma membrane. Unique features of this structure are the presence of inositol, only found in eukaryotic cells and some archaebacteria and the presence of a unique type of lipids, the mycolates and corynomycolates. An updated perusal of the published literature agreed that the mycobacteria and corynebacteria envelopes share some gross structural features and similar cell wall architecture7.
Arabinogalactan (AG) and lipoarabinomannan (LAM) which are arabinan-containing polysaccharides are key components of the cell wall of Corynebacterineae, which comprise corynebacteria, norcardia and mycobacteria8.
However, fundamental differences between these two genus are found in the composition of these features. Analysis by quantitative sugar and glycosyl linkage showed that C. glutamicum have a tiny version of LAM, called Cg-LAM9.
Mycolyl-transferases are a clan of proteins that are especially present in the CMN genera (Corynebacterium, Mycobacterium and Nocardia), mycolyl-transferases are responsible for cell wall components synthesis10. The cell walls of the members of CMN genera organisms are composed of linked peptidoglycan and polysaccharide-mycolate complex and are attributed by the existence of mycolic acid on their surface11.
The micro-organisms belonging to CMN genera are gathered together as a group on the ground of some aspects including complicated cell wall components, type and presence of mycolic acids, adjuvant activity, presence of cord factor, sulfo-lipids, iron-chelating compounds, polyphosphate and serological cross-reactivity.
Conceptually, the mycobacterial and corynebacterial envelopes are composed of two coats of complex structure: an outer coat and an inner coat. Indeed, a complete cell-wall lipid bilayer is observed even in a C. glutamicum mutant whose cAGM content is further reduced to half the wild-type value. The current models of the corynebacterial envelope structure of the cell-wall envelope6,12 proposed that the cell-wall lipid bilayer is mostly formed essentially of small free lipids, predicting a 2-3 nm for the thickness of the bilayer-associated electron-transparent layer. Thus, the cell-wall lipid bilayer cannot account for the observed thickness of the corynebacterial Electro Transparent Layer (ETL) structure. This observation also reopens earlier questions raised about the nature of the ETL in mycobacteria4,6.
Although, many studies concerned with physiological and gene cloning aspects of Escherichia coli13 and Corynebacterium were executed14,15 whether expressed in Escherichia coli16,17 or C. glutamicum5, but genes coding of glycosyl-transferases of C. glutamicum were not been among them.
As C. glutamicum and M. tuberculosis share a similar cell wall architecture, enabling the utilization of C. glutamicum as a model for the identification and study of, otherwise; essential, mycobacterial genes involved in lipomannan (LM) and lipoarabinomannan (LAM) biosynthesis7. Understanding the genes coding of glycosyl-transferases of C. glutamicum would help among other things to develop antibiotics against M. tuberculosis. The aim of the present work is to identify C. glutamicum genes encoding for glycosyl-transferases enzymes activity.
MATERIALS AND METHODS
Study area: This study was conducted from March, 2000 to December, 2003 at Institut de Génétique et Microbiologie (IGM), Université Paris XI-Sud, France and from March, 2019-2020 at Colleges of Applied Medical Sciences, Medical Rehabilitation Sciences, Pharmacy at Taibah University, Saudi Arabia.
Bacterial strains and growth conditions: Bacterial strains and vectors used throughout the work are listed in Table 1 and 2. Growing of Corynebacterium strains was carried out aerobically (250 rpm) at 34°C, either in Brain Heart Infusion (BHI; Difco) (Fisher Scientific, USA) rich medium or in MCGC minimal medium as described by Von der Osten et al.18, except that citrate (used as a chelating agent) was replaced by deferoxamine. Following the growth of Corynebacterium strains was done by measuring the optical density at 570 nm (OD570) in a DU 7400 Beckman spectrophotometer (Irvine, USA). Kanamycin and chloramphenicol were added to medium when needed at concentrations of 25 and 15 μg mL–1, respectively. Growing of Escherichia coli strains was carried out aerobically at 37°C in Luria Bertani (LB) medium (Difco) (Fisher Scientific, USA) . Following the growth of E. coli strains was done by measuring the optical density at 600 nm (OD600). Ampicillin, kanamycin and chloramphenicol were added to medium when needed at concentrations of 100, 25 and 30 μg mL–1, respectively. All chemicals used were purchased from Sigma (Sigma-Aldrich, Missouri, USA).
DNA manipulations: Basic molecular biology methods used were done as described by Sambrook et al.23. Enzymes were purchased from Promega (Madison, USA), Biolab (Middlebury, USA) and Boehringer Mannheim (Rotkreuz, Switzerland). Extraction of plasmidic DNA was done by using the Wizard kit from Promega (Madison, USA).
| Table 1: | Bacterial strains used in this study |
| ATCC: American type culture collection, Rockville, USA | |
| Table 2: | Plasmids used or constructed in this study |
| Ampr: Resistance for the ampicilline, Kanr: Resistance for the kanamycine | |
Isolation of DNA fragments from agarose gels was done by using the Jetsorb kit (Genomed, Florida, USA). Extraction of chromosomal DNA of C. glutamicum was done by the method described by Ausubel et al.24. Transformation of Corynebacterium strains was done by electroporation25. Southern blotting technique was used for checking the integration of the introduced DNA into the chromosome DNA of Corynebacterium26. The DNA probes used were prepared and labeled non-radioactively using the DIG DNA Labeling and Detection kit (Roche, Basel, Switzerland). The colonies held for more studies were purified on selective solid medium. Storing of strains was done by picking up 1 mL of liquid medium of stationary-phase cultures inoculated with a single colony; mixing it with 1 mL of 80% glycerol, storing at -20°C.
Determination and analysis of the nucleotidic sequences: Nucleic acid sequences were determined by ESGS society. The method used for nucleic acid sequences determination was of Sanger et al.27. Analysis of the nucleotidic sequences was carried out using the software GeneJockey (Sigma-Aldrich, Missouri, USA), DNA Strider (LabStrider), Blast (NCBI) and CLUSTAL W (Clustal Omega).
PCR amplification of a pimA gene fragment: A pair of oligo-nucleotide mixtures (MT1: [GACGTBCTNCAYACGARCC] and MT2: [CCATGGCYTCKACSAGVACGATGCC] was used for PCR amplification of the chromosomal DNA of the C. glutamicum sp. 2262 strain. The reaction mixture for PCR amplification contained 2.5 U of thermostable DNA polymerase (AmpliTaqGold, Perkin-Elmer), 30 ng of genomic DNA, 0.2 mM each deoxynucleotide triphosphates (Promega) (Madison, USA), 2 mM MgCl2 , 1×Ampli Taq Buffer (Thermo Fisher Scientific, USA) in a final volume of 50 μL and 0.5 μM each of both primers. The first cycle (10 min at 94°C) followed by 35 identical cycles (1 min at 94°C, 1 min at 50°C and 1 min at 72°C) then followed by a final elongation cycle (5 min at 72°C).
RESULTS AND DISCUSSION
Identification of target glycosyl transferases: The NCBI and Sanger Institute databases were searched for “glycosyl transferases” that were common both to Mycobacterium tuberculosis and Corynebacterium difteriae. This search identified a conspicuous protein, a cellobiosyl-diphosphoprenyl alpha-mannosyl transferase hereafter named "PimA" (pimA). PimA-like sequences are represented in several instances in the taxonomically related M. tuberculosis, Mycobacterium leprae and C. diphtheriae. Similar sequence to PimA was also present in the actinomycete Streptomyces coelicolor and in the extremophile archeons Pyrococcus horikoshii, Aeropyrum pernix and Pyrococcus abyssi. In the same trend, Berg et al.28 showed that glycosyltransferases (Gts) of M. tuberculosis have orthologs in prokaryotes and eukaryotes.
Amplification of an internal pimA ORF fragment by PCR: Alignments in Sanger Institute of the presumed PimA proteins of C. diphteriae (contig 358- 373623:374684), M. tuberculosis (A70571) and M. leprae (CAB09632.1) showed highly conserved regions which permitted the design of mixtures of oligo-nucleotides pairs intended to PCR amplification of a pimA gene fragment devoid of the sequences needed to encode the PimA carboxy-terminal and amino-terminal regions. The chosen pair of oligonucleotide mixtures can be schematized as MT1 (GACGTBCTNCAYACGARCC) and MT2 (CCATGGCYTCKACSAGVACGATGCC) with respect of the codon preferences observed in Corynebacterium glutamicum and related species29. In theory, the oligonucleotides MT1 and MT2 could amplify a DNA band of about 550 bp located in the central portion of the pimA gene which exclude the amino-terminal and carboxy-terminal ends of the gene ORF. An amplified DNA band of the expected size (0.55 kpb) was obtained from the strain Corynebacterium sp. 226230, which will be hereafter referred to as “C. glutamicum Or2262” in basis of its high DNA sequence identity to C. glutamicum ATCC13032 and in the presence on it of corynebacterial LAM. This fragment was cloned into PCR 2.1-TOPO vector (Invitrogen®, California, USA). Transformants of the expected structure were identified by sequencing the insert ends using the oligonucleotides primers F-20 (M13 Forward-primer; GTAAAACGACGGCCAGT) and REV (M13 Reverse-primer; CAGGAAACAGCTATGACC). One of such plasmids pSG1 contained an insert highly homologous to the pimA genes of the mycobacteria and of C. diphteriae. Figure 1 showed amplification and cloning of a glycosyl transferase fragment C. glutamicum Or2262. This suggested that pSG1 insert correspond to a fragment of a Corynebacterium sp. 2262 gene that belongs to the pimA family.
Cloning of the internal fragment of pimA gene in C. glutamicum Or2262: The plasmid pSG1 carries the gene aphII, encoding for an aminoglycoside-phosphotransferase which determined resistance to kanamycin (KmR phenotype). Since, pSG1 is not replicative in C. glutamicum, most Kmr transformants should arose by homologous recombination with the cognate host chromosomal region.
| Fig. 1: | Amplification and cloning of a glycosyl transferase fragment Corynebacterium glutamicum Or2262 |
If plasmid pSG1 contains as expected, an internal pimA gene fragment, integration at the bacterial genome by a single homologous recombination event at the identical wild-type pimA gene of C. glutamicum Or2262 should lead to host pimA inactivation. Figure 2 showed interruption of pimA gene of fragment C. glutamicum. It refer hereafter to the pimA mutant allele further studied as pimA1. It also isolated insertions of pSG1 in C. glutamicum ATCC13032 RES167, one of is denominated pimA2. The transformability of C. glutamicum Or2262 (Table 3) is 18.0 times lower than that of C. glutamicum ATCC13032 RES167 as evaluated with the highly transformable plasmid pCGL0243, replicative in C. glutamicum. The transformability of C. glutamicum Or2262 with the integrative plasmid pSG1 is 71.5 times lower than that of the strain C. glutamicum ATCC13032 RES167, despite the fact that the sequence homology that determines host recombinational integration of pSG1 is perfect in the case of the former, which suggested that the limiting step in the efficiency of integrative transformation of C. glutamicum Or2262 is not diffusion of the plasmid through the bacterial envelope, but an efficiency of plasmid-host recombination lower than that of C. glutamicum ATCC13032 RES167. In corynebacteria, very high voltage(up to 40 kV cm–1) electro-transformation has been achieved for intact C. glutamicum cells, though temperature and pressure effects limited the transformant yield to 3×103/μg of DNA31. Bonamy et al.25 reported successful and efficient electro-transformation of plasmid DNA into intact cells of nine corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacteriuum flavum, C. glutamicum and Corynebacterium melassecola. In optimal conditions, more than 107 transformants per/μg of DNA could be obtained.
Accepted FMTR (Average frequency of replicative transformation) values were 3.84×10–5 for C. glutamicum ATCC13032 RES167 and 4.6×10–5 for C. glutamicum Or2262.
Isolation of the affected mutant in the pimA gene of C. glutamicum: The partial sequence of the plasmid containing the fragment of pSG1 and its insertion site directly indicated that the insertion of pSG1 defined mutations in the pimA gene. The genomic DNA of three pSG1 insertions in C. glutamicum Or2262 were digested with BamHI, EcoRV and HindIII and then were subjected to hybridization analysis with pSG1 probe. Analysis results was found to be consistent with integration by homologous recombination of plasmid pSG1 in the host pimA gene. The integrated of pimA allele and the prediction of hybridization bands are shown in Fig. 3a-b, respectively. Southern hybridization of pimA mutants DNA of C. glutamicum is shown in Fig. 4.
| Fig. 2: | Interruption of pimA gene of fragment Corynebacterium glutamicum |
| Fig. 3(a-b): | (a) Integrated pimA allele and (b) Prediction of hybridization bands |
A similar analysis was done by HindIII digestion of the DNA extracted from pSG1 transformant in the C. glutamicum ATCC13032 RES167 host probed by pCGL3182 revealed also the existence of an insertion in the pimA region (Fig. 4: channel 1). Many scientists successfully achieved mutants constructions of CMN genera. Tatituri et al.32 described the disruption of PimB probable orthologue Cg-pimB and the chemical analysis of glycolipids and lipoglycans isolated from wild type C. glutamicum and the C. glutamicum::pimB mutant. Mishra et al.33 also described a structural characterization and functional properties of a lipomannan variant isolated from a C. glutamicum pimB mutant. The construction of a pimA conditional mutant of M. smegmatis was also achieved by Korduláková et al.34. To determine whether PimA is an essential enzyme of mycobacteria, Korduláková et al.34 constructed a pimA conditional mutant of M. smegmatis and showed that the ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature- sensitive rescue plasmid.
| Fig. 4: | Southern hybridization of pimA mutants DNA of C. glutamicum |
| Table 3: | Characteristics of the integrative transformation in the pimA region |
a, b, c and d refer to preparations of competent cells by electrotransformation, FTR: Frequency of replicative transformation of the batch (transformability with pCGL0243 or pCGL0609), FTI: Frequency of integrative transformation of the batch (transformability with pSG1, pCGL3181 or pCGL3182), FNTI: Normalized frequency of integrative transformation [FTI * (FTR/FMTR)] | |
The sequence analysis of plasmid pSG1 indicated that the insertion of the pimA ORF internal fragment is in line with amino-terminal extremity of the lacZ ORF fragment of the vector pCR2.1-TOPO (Invitrogen®, California, USA). Although, the lacZ gene promoter is not functional in C. glutamicum35, but our results showed that there are two oriented promoters are present upstream in the same direction of lacZ gene. These promoters are promoters of aphA2 and bla genes of the vector which are well expressed in C. glutamicum.
The implications of previous results include opening the door for cloning of complete pimA gene of C. glutamicum. Cloning of complete pimA gene of C. glutamicum should make we able to analysis the whole pgsA-htrB-pimA region encoding for glycosyl-transferase enzyme activity of the species C. glutamicum. But studying the phenotypes of pimA, htrB and pgsA insertions is mandatory to understand the role and functionality of these genes.
CONCLUSION
PimA-like similar sequences were found to be widespread in many taxonomically related species to Corynebacterium glutamicum, which reflects the importance of glycosyl-transferases enzymes activity in such species. Complete pimA gene of C. glutamicum should be cloned to be able to analysis the whole pgsA-htrB-pimA region. Studying the phenotypes of pimA, htrB and pgsA insertions is mandatory to understand the role and functionality of these genes.
SIGNIFICANCE STATEMENTS
This study discovered that cloning of an internal fragment of pimA gene coding glycosyl-transferase of Corynebacterium glutamicum is successfully achievable. The genes encoding for glycosyl-transferases enzymes activity was identified. This study will help the researchers to understand the cell wall structure of CMN genera (Corynebacterium, Mycobacterium and Nocardia) which in turn would help among others in developing more functional antibiotics against Mycobacterium tuberculosis.
ACKNOWLEDGMENT
We are grateful to Dr. Gérard Leblon, Dr. Oscar Reys, for valuable help, advices and discussions throughout the work, to Dr. A. Guyonvarch for his assistance in primer designing, to Dr. J. Kalinowski for the gift of the Corynebacterium glutamicum RES167 strain.