Beukes M, Hastings J W
School of Molecular and Cellular Biosciences, University of Natal, Pietermaritzburg, Scottsville 3209, South Africa.
Appl Environ Microbiol. 2001 Sep;67(9):3888-96. doi: 10.1128/AEM.67.9.3888-3896.2001.
Streptococcus milleri NMSCC 061 produces an endopeptidase, millericin B, which hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. The nucleotide sequence of a 4.9-kb chromosomal region showed three open reading frames (ORFs) and a putative tRNA(Leu) sequence. The three ORFs encode a millericin B preprotein (MilB), a putative immunity protein (MilF), and a putative transporter protein (MilT). The milB gene encodes a 277-amino-acid preprotein with an 18-amino-acid signal peptide with a consensus IIGG cleavage motif. The predicted protein encoded by milT is homologous to ABC (ATP-binding cassette) transporters of several bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. These similarities strongly suggest that the milT gene product is involved in the translocation of millericin B. The gene milF encodes a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of Staphylococcus aureus, which are involved in the addition of glycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell wall mucopeptide of S. milleri NMSCC 061(resistant to lysis by millericin B) and S. milleri NMSCC 051(sensitive) showed a single amino acid difference. Serial growth of S. milleri NMSCC 051 in a cell wall minimal medium containing an increased concentration of leucine resulted in the in vivo substitution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of S. milleri NMSCC 051 (sensitive) that contained an amino acid substitution (leucine for threonine) within its peptidoglycan cross bridge showed partial susceptibility to millericin B. The putative tRNA(Leu) sequence located upstream of milB may be a cell wall-specific tRNA and could together with the milF protein, play a potential role in the addition of leucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.
米勒链球菌NMSCC 061产生一种内肽酶，即米勒菌素B，它能水解易感细胞壁肽聚糖的肽部分。一个4.9kb染色体区域的核苷酸序列显示有三个开放阅读框（ORF）和一个假定的tRNA(Leu)序列。这三个ORF分别编码一个米勒菌素B前体蛋白（MilB）、一个假定的免疫蛋白（MilF）和一个假定的转运蛋白（MilT）。milB基因编码一个277个氨基酸的前体蛋白，带有一个18个氨基酸的信号肽，具有一致的IIGG切割基序。由milT预测的蛋白质与几种细菌素系统的ABC（ATP结合盒）转运蛋白以及与大肠杆菌溶血素A的信号序列非依赖性输出有关的蛋白质同源。这些相似性强烈表明milT基因产物参与了米勒菌素B的转运。milF基因编码一个302个氨基酸的蛋白质，它与金黄色葡萄球菌的FemA和FemB蛋白相似，后者参与将甘氨酸添加到五肽肽聚糖前体中。对米勒链球菌NMSCC 061（对米勒菌素B裂解有抗性）和NMSCC 051（敏感）的细胞壁粘肽进行比较显示有一个氨基酸差异。米勒链球菌NMSCC 051在含有增加浓度亮氨酸的细胞壁基本培养基中连续传代培养，导致细胞壁粘肽中体内亮氨酸替代苏氨酸。米勒链球菌NMSCC 051（敏感）的一个细胞壁变体，其肽聚糖交联桥内含有氨基酸替代（亮氨酸替代苏氨酸）显示出对米勒菌素B的部分敏感性。位于milB上游的假定tRNA(Leu)序列可能是一种细胞壁特异性tRNA，并且可能与milF蛋白一起，在将亮氨酸添加到五肽肽聚糖前体中发挥潜在作用，从而有助于产生菌对米勒菌素B进行自我保护。
