vanY Gene Research Articles

Endophytic Bacterium Flexivirga meconopsidis sp. nov. with Plant Growth-Promoting Function, Isolated from the Seeds of Meconopsis integrifolia.

Strain Q11T of an irregular coccoid Gram-positive bacterium, aerobic and non-motile, was isolated from Meconopsis integrifolia seeds. Strain Q11T grew optimally in 1% (w/v) NaCl, pH 7, at 30 °C. Strain Q11T is most closely related to Flexivirga, as evidenced by 16S rRNA gene analysis, and shares the highest similarity with Flexivirga aerilata ID2601ST (99.24%). Based on genome sequence analysis, the average nucleotide identity and digital DNA-DNA hybridization values of strains Q11T and D2601ST were 88.82% and 36.20%, respectively. Additionally, strain Q11T showed the abilities of nitrogen fixation and indole acetic acid production and was shown to promote maize growth under laboratory conditions. Its genome contains antibiotic resistance genes (the vanY gene in the vanB cluster and the vanW gene in the vanI cluster) and extreme environment tolerance genes (ectoine biosynthetic gene cluster). Shotgun proteomics also detected antibiotic resistance proteins (class A beta-lactamases, D-alanine ligase family proteins) and proteins that improve plant cold tolerance (multispecies cold shock proteins). Strain Q11T was determined to be a novel species of the genus Flexivirga, for which the name Flexivirga meconopsidis sp. nov. is proposed. The strain type is Q11T (GDMCC 1.3002T = JCM 36020 T).

Study of the internal mechanism of L-glutamate for improving the survival rate of Lactiplantibacillus plantarum LIP-1 after freeze-drying

Lactic acid bacteria are important fermentation strains, and improving their freeze-drying survival rate (FDSR) has become a hot research topic in recent years. In this research, we found that adding 0.1 g/L L-glutamate to culture medium improved the FDSR of four strains of L. plantarum. Using L. plantarum LIP-1 as an example, we explored the internal mechanism of this phenomenon. L-glutamate had this effect through two metabolic pathways. First, it made the strain grow rapidly and produce more acid, resulting in a rapid decline of pH in the culture environment. The acidic environment led to the upregulation of the dnaK gene and secG gene expression, increased DnaK protein levels, promoted its anchoring effect on the cell membrane, and enhanced the integrity of the cell membrane. Second, it upregulated the expression of the murL, murD, and vanY genes, increased peptidoglycan synthesis, and increased cell wall integrity. In sum, the strain obtained a higher FDSR by adjusting the cell membrane and cell wall structure using L-glutamate. This study provides a new method for improving the FDSR of lactic acid bacteria.

Heteroresistance to vancomycin and novel point mutations in Tn 1546 of Enterococcus faecium ATCC 51559

A clinical strain of Enterococcus faecium ATCC 51559 exhibits heteroresistance, i.e. a high level of resistance to vancomycin (minimum inhibitory concentration (MIC) > 256 μg/mL) by broth dilution but sensitivity to vancomycin by Etest (MIC = 1.8 μg/mL). Three variants of this strain, EF1, EF2 and EF3, exhibit high levels of resistance to vancomycin both by broth dilution and Etest assays. The four strains were used to study heteroresistance by pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequence analysis of a partial region of the van operon. Minor differences between SalI and SmaI restriction profiles of the variants and the parental strain were observed by PFGE analysis. PCR analysis confirmed the presence of the vancomycin resistance marker vanA (0.73 kb) and a larger than expected amplicon (8.2 kb vs. 6.7 kb) of the van operon in all the strains. The 8.2 kb van operon was cloned for EcoRI RFLP and sequence analysis. All of the clones exhibited distinctly different RFLP profiles when grown in the presence of kanamycin or vancomycin + kanamycin. The presence of these antibiotics during overnight growth of EF1 on plates also resulted in altered SalI PFGE profiles. Sequence analysis of the van operon clones revealed a 1.5 kb IS 1251-like insertion element between the vanS and vanH genes in all the strains. Several novel point mutations in the vanR, vanS, vanH, vanA, vanX and vanY genes were also discovered. Some of these mutations were present in the parental strain only and included base substitutions T → C, A → G, T → A and T → C at nucleotide positions 4202, 4597, 4763 and 6207 of Tn 1546, resulting in amino acid replacements I76 → T and K208 → E of vanR, S19 → T of vanS and L64 → P of vanH genes, respectively. We believe that these are responsible for the observed heteroresistance. The present study clearly shows how independent novel mutations can give rise to polymorphism, heteroresistance and clonal diversity among vancomycin-resistant enterococci strains as a result of continuous exposure to antibiotics.

Mechanism of VanB Phenotype in Vancomycin-Resistant Enterococci carrying vanA gene

Recently, vancomycin-resistant enterococci (VRE) with the vanA genotype that are susceptible to teicoplanin have been described in Japan, Taiwan, and Korea. The investigators suggested three point mutations in the putative sensor domain of vanS or impairment of accessory proteins VanY and VanZ as an explanation for the VanB phenotype-vanA genotype VRE. In this study, we analyzed Tn1546-like elements to determine the molecular mechanisms responsible for the impaired glycopeptide resistance of clinical VRE isolates with VanB phenotype-vanA genotype from Korea. From 2001 to 2004, 28 clinical isolates of Enterococcus faecium with VanB phenotypevanA genotype were collected from 8 different university hospitals in diverse geographic areas in Korea. For structural analysis of Tn1546-like elements, PCR amplifications for internal regions of Tn1546 were performed. The purified PCR products were directly sequenced with an ABI Prism 3100 DNA sequencer. The sequence data of the vanS regulatory gene revealed that none of the isolates had any point mutations in this gene. All 28 isolates had a complete or incomplete deletion of vanY gene. Of these, 13 strains represented a complete deletion of vanZ, and 2 strains showed the deletion of nucleotides near the end point of vanX. The mechanism of VanB phenotype-vanA genotype in VRE isolates from Korea is not point mutations of vanS but the rearrangements of vanX, vanY and vanZ.

Molecular Analysis of Tn 1546 in vanA -Containing Enterococcus spp. Isolated from Humans and Poultry

The genes encoding the VanA type of vancomycin resistance in enterococci are located on elements related to Tn1546 (1). Heterogeneity of Tn1546 in Enterococcus faecium has been previously reported (3, 4, 6, 9, 10, 12, 14, 15, 16) and includes deletions, insertions, and mutations. In this study, Tn1546 elements from 43 vanA-containing isolates of different enterococcal species with diverse pulsed-field gel electrophoresis (PFGE) patterns (criteria of Tenover et al. [13]) were analyzed: 30 E. faecium isolates (18 from chicken feces or products and 12 from human fecal samples) showing 25 unrelated PFGE patterns, 7 Enterococcus durans isolates with two unrelated PFGE patterns, (chicken feces or products), 5 Enterococcus hirae isolates (4 from chicken feces or products and 1 from a human fecal sample known to have an indistinguishable or a closely related PFGE pattern), and 1 Enterococcus faecalis isolate (from a chicken product). PCR products were obtained from all isolates for the seven genes of Tn1546 (vanR, vanS, vanH, vanY [10], vanA [16], vanX [11], and vanZ [8]); IS1216V-related sequences (6) were also demonstrated by PCR for all 43 isolates. When the vanXY (9) region was amplified, the expected 1,947-bp fragment was obtained with 36 of the 43 isolates; however, in all 5 E. hirae isolates and in 2 E. faecium isolates (both from chickens, with different PFGE patterns) the fragment amplified was longer than expected. Results of hybridization of vanXY PCR products with an IS1216V probe indicated that IS1216V was located within the vanXY region in these seven isolates and outside this region in the other isolates. An IS1216V-like sequence was first described within the intergenic vanXY region (5) and later both within and outside Tn1546 (3, 6); disruption of vanS by IS1216V has been found in a clinical E. faecium isolate (2). Despite these reports for E. faecium, this is the first time that IS1216V has been reported within the vanXY region of E. hirae. The restriction of the IS1216V-vanXY association to E. hirae and two E. faecium strains isolated from chickens could suggest interspecies transmission of these transposons in animal gastrointestinal tracts. IS1251 sequences (6) were detected in 18 E. faecium isolates (13 from chickens and 5 from humans; 15 unrelated PFGE patterns) but not in the other species tested. Analysis of vanSH (9) amplicons and hybridization showed that IS1251 was not included in this region. IS1251 has been previously found in the vanSH intergenic region and at other sites in E. faecium (4, 6). IS1476, first found in the vanY gene of an E. faecium isolate (9), was not detected in our strains nor in two other studies (6, 15). Recently, a 1-bp difference in vanX was found at position 8234, with either a G (G type) or a T (T type) (6). All our vanA isolates, except one (E. faecium from ground chicken), belonged to the G type as determined by DdeI digestion of the vanX gene PCR fragment (315 bp) (6). The G type has been associated with poultry, and the T type has been associated with porcine E. faecium isolates (3, 7). Both types have been found among isolates from humans in different countries (7), although we found only the G type among vancomycin-resistant enterococci isolated from humans in Spain. In conclusion, vanA-containing E. faecium, E. faecalis, E. hirae, and E. durans strains of human and animal origins were found to contain similar genetic arrangements of the vanA gene cluster, suggesting either horizontal transfer, the existence of a common reservoir, or a predilection for insertion of certain elements at specific sites.

Internal size variations in Tn 1546-like elements due to the presence of IS 1216V

In this study, internal size variations in the VanA gene cluster Tn 1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn 1546, revealed that these variations were due to the presence of the IS sequence, IS 1216V. This IS sequence has previously been found integrated in Tn 1546. Integration of the IS 1216V element created both deletions and a duplication in a non-essential region of Tn 1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.

Internal size variations in Tn1546-like elements due to the presence of IS1216V.

In this study, internal size variations in the VanA gene cluster Tn1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn1546, revealed that these variations were due to the presence of the IS sequence, IS1216V. This IS sequence has previously been found integrated in Tn1546. Integration of the IS1216V element created both deletions and a duplication in a non-essential region of Tn1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.

Identification and characterization of IS1476, an insertion sequence-like element that disrupts VanY function in a vancomycin-resistant Enterococcus faecium strain

The vanY gene of vancomycin-resistant enterococci encodes a D,D-carboxypeptidase. By using a PCR detection strategy, a VanA Enterococcus faecium clinical isolate was found to have an insertion sequence (IS)-like element designated IS1476 in vanY. The activity of the VanY D,D-carboxypeptidase in this isolate was decreased in a fluorometric fluoraldehyde o-phthalaldehyde assay with diacetyl-L-Lys-D-Ala-D-Ala as the substrate. This, to our knowledge, is the first report of an IS-like element in a vancomycin resistance gene.

Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in VanA isolates

Vancomycin resistance in enterococci is an emerging therapeutic problem. Resistance is not always detected by standard microbiological methods. Oligonucleotide primers for PCR were designed to target amplification of defined regions of genes of the vanA cluster, as well as vanB and vanC1. These primers correctly identified 30 vancomycin-resistant isolates tested (17 VanA, 7 VanB, and 6 Enterococcus gallinarum). No amplification was observed with Enterococcus casseliflavus or vancomycin-susceptible strains. Using PCR and Southern blotting, we found that all 17 VanA isolates had orf-1, orf-2, vanR, vanS, vanH, vanA, and vanY genes in the same sequence and that the intergenic distances in the vanR-vanA segments were the same. The described methods should be applicable to the rapid detection of the different vancomycin resistance genotypes in enterococci.

Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors

The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.

Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147

Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible d,dcarboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.

Characterization of vanY, a DD-carboxypeptidase from vancomycin-resistant Enterococcus faecium BM4147.

VanY is a protein with a molecular mass of 34.8 kDa encoded by vanY, a member of the high-level vancomycin resistance gene cluster found on plasmid pIP816 in Enterococcus faecium BM4147. Extracts from Escherichia coli JM83 bearing plasmid pAT383, which contains the vanY gene, were examined for enzymatic hydrolysis of peptidoglycan precursors. VanY was associated with the cell membranes and cleaved the C-terminal D-alanine residue of UDP-muramyl-pentapeptide but did not display transpeptidase or beta-lactamase activities. The DD-carboxypeptidase activity was not inhibited by beta-lactam antibiotics. VanY released the C-terminal D-hydroxy acid from depsipeptides produced by the vancomycin resistance protein VanA. These results demonstrate that VanY should contribute in vivo to the hydrolysis of both the D-alanyl-D-alanine- and the depsipeptide-containing peptidoglycan precursors.