图1 CR-HMKP K2-ST375中前噬菌体整合位置及Prophage-诱导引物扩增区域示意图 四种不同颜色分别代表四个前噬菌体。F1 - F4分别是Prophage-1 - Prophage-4诱导鉴定的正向引物；R1 - R4分别是Prophage-1 - Prophage-4诱导鉴定的反向引物

Fig. 1 Schematic diagram of the integration sites and Prophage-induced primer amplification region in CR-HMKP K2-ST375 The four different colors indicate the four prophages, respectively. F1 - F4 are the forward primers for Prophage-1 - Prophage-4 induced identification, respectively; R1 - R4 are the reverse primers for Prophage-1 - Prophage-4 induced identification, respectively

图2 Prophage-4诱导及环化结构验证 A：丝裂霉素C诱导Prophage-4：M：DNA marker DL2000；1泳道：使用Prophage-4-诱导引物扩增菌上清；2泳道：使用管家基因phoE引物扩增菌上清；3泳道：阴性对照；B：Prophage-4环化鉴定：M：DNA marker DL2000；1泳道使用Prophage-4-环化引物扩增菌上清；2泳道使用管家基因phoE引物扩增菌上清；3泳道：阴性对照

Fig. 2 Induction and verification of circularization structure of Prophage-4 A: Prophage-4 induction by mitomycin C; M: DNA marker DL2000; lane 1: the Prophage-4-induction primers were used to amplify the bacterial supernatant; lane 2: the primer of phoE were used to amplify the bacterial supernatant; lane 3: negative control. B: Identification of circularization structure of Prophage-4; M: DNA marker DL2000; lane 1: the Prophage-4-cyclization primer were used to amplify the bacterial supernatant; lane 2: the primer of phoE were used to amplify the bacterial supernatant; lane 3: negative control

图3 Prophage-4环化结构形成示意图 第一个圈图表示整合有Prophage-4的Kp0179染色体，其中，蓝色为Prophage-4基因组，红色和黄色分表代表40 bp的同源重复序列HR1和HR2；其相邻的attL为左侧附着位点，attR为右侧附着位点。中间圈图表示Kp0179染色体上与Prophage-4基因组存在40 bp同源重复序列。右侧圈图表示Prophage-4从Kp0179染色体上剪切下来形成phage-4，绿色大圆圈表示剪切后的Kp0179染色体。虚线框中为Prophage-4的环化引物扩增区域

Fig. 3 Schematic diagram of Prophage-4 circular structure formation The first diagram shows the integration of Prophage-4 into the Kp0179 chromosome, where blue indicates the Prophage-4 genome, red and yellow respectively indicate the 40 bp homologous repeats sequences HR1 and HR2; the adjacent attL is the left attachment site, and attR is the right attachment site. The middle diagram shows the Kp0179 chromosome with the 40 bp homologous repeats sequences present in the Prophage-4 genome. The diagram on the right shows Prophage-4 being excised from the Kp0179 chromosome to form phage-4, with the large green circle representing the Kp0179 chromosome after excision. In the dotted box is an amplified region of the cyclization primer for Prophage-4

图4 CR-HMKP K2-ST375染色体中Prophage-4遗传结构 从内至外：第一圈为Prophage-4的基因组长度；第二圈为GC含量；第三圈为GC偏移量；最外圈代表Prophage-4的编码基因

Fig. 4 Genetic structure of Prophage-4 in the chromosome of CR-HMKP K2-ST375 From the inside to the outside: the first layer refers to the genome length of Prophage-4; the second layer refers to the GC content; the third layer refers to the GC skew; and the outermost layer refers to the coding genes of Prophage-4

图5 SignalP 4.1预测内溶素蛋白的信号肽 曲线的横坐标是序列，纵坐标是分值。紫、绿、蓝3种颜色分别表示剪切位点分值（C值）、信号肽分值（S值）、C值和S值的综合计分值（Y值）；文本输出结果中，“NO”表示内溶素蛋白不包含信号肽序列

Fig. 5 Prediction of signal peptides in endolysin by SignalP 4.1 The horizontal axis of the curve indicates the sequence, and the vertical axis indicates the score. Purple, green, and blue colors indicate the cleavage site score（C-score）, signal peptide score（S-score）, and the combined score of C-score and S-score（Y-score）, respectively. In the text output results, “NO” indicates that the endolysin does not contain a signal peptide sequence

图6 DeepTMHMM预测内溶素蛋白的跨膜区 曲线的横坐标是序列，纵坐标是概率。蓝色表示该蛋白在膜外的概率，黄色表示信号肽的概率

Fig. 6 Transmembrane domains prediction of endolysin by DeepTMHMM The horizontal axis of the curve represents the sequence, and the vertical axis represents the probabilitity. Blue indicates the probability of the protein being outside the membrane, while yellow indicates the probability of signal peptides

图7 NetPhos-3.1分析内溶素蛋白的磷酸化位点 横坐标是序列，纵坐标是磷酸化概率。红、绿、蓝、粉4种颜色分别表示丝氨酸（Serine）、苏氨酸（Threonine）、酪氨酸（Tyrosine）以及阈值（Threshold）；当阈值大于0.5表示该位置氨基酸为磷酸化位点

Fig. 7 Analysis of the phosphorylation sites in endolysin using NetPhos-3.1 The horizontal axis refers to the sequence, while the vertical axis refers to the probability of phosphorylation. The four colors, red, green, blue, and pink, indicate serine, threonine, tyrosine, and the threshold, respectively. When the threshold exceeds 0.5, it indicates that the amino acid at that position is a phosphorylation site

图8 Phyre2预测内溶素蛋白的二级结构 第一行为序列位置：黄色为极性氨基酸：A（丙氨酸）、S（丝氨酸）、T（苏氨酸）、G（甘氨酸）及P（脯氨酸）；绿色为疏水性氨基酸：M（蛋氨酸）、I（异亮氨酸）、L（亮氨酸）和V（缬氨酸）；红色为荷电性氨基酸：K（赖氨酸）、R（精氨酸）、E（谷氨酸）、N（天冬酰胺）、D（天冬氨酸）、H（组氨酸）及Q（谷氨酰胺）；紫色为芳香族的氨基酸：W（色氨酸）、Y（酪氨酸）、F（苯丙氨酸）及C（半胱氨酸）；第二行为二级结构，绿色为α螺旋，蓝色为β折叠。第三行和第四行为置信度评估：红色表示高置信度9，蓝色为低置信度0，中间共划分10个等级

Fig. 8 Prediction of the secondary structure of the endolysin using Phyre2 The first line indicates the sequence positions: Yellow indicates polar amino acids: A（Alanine）, S（Serine）, T（Threonine）, G（Glycine）, and P（Proline）; green indicates hydrophobic amino acids: M（Methionine）, I（Isoleucine）, L（Leucine）, and V（Valine）; red indicates charged amino acids: K（Lysine）, R（Arginine）, E（Glutamic acid）, N（Asparagine）, D（Aspartic acid）, H（Histidine）, and Q（Glutamine）; purple indicates aromatic amino acids: W（Tryptophan）, Y（Tyrosine）, F（Phenylalanine）, and C（Cysteine）. The second line indicates the secondary structure, with green for α-helices and blue for β-sheets. The third and fourth lines are for confidence assessment: red indicates high confidence 9, blue indicates low confidence 0, with a total of 10 levels divided in between

图9 SWISS-MODEL预测内溶素蛋白的三维结构 螺旋表示α螺旋，片状箭头表示β折叠，线条表示无规卷曲

Fig. 9 Prediction the three-dimensional structure of the endolysin by SWISS-MODEL Helices indicate alpha helices, sheet-like arrows indicate beta sheets, and lines indicate random coils

图10 CD Search预测内溶素蛋白的保守结构域 第一行为序列位置；蓝色区域表示该氨基酸序列与溶菌酶超家族蛋白质完全匹配，其中深蓝色部分与endolysin R21-like 具有较高的匹配度；蓝色三角形表示该氨基酸为已注释的功能位点

Fig. 10 Conserved structural domains prediction of endolysin by CD Search The first line indicates the sequence position; the blue region indicates that this amino acid sequence completely matches with the lysozyme superfamily proteins, where the dark blue parts have a higher degree of match with endolysin R21-like; the blue triangles indicate that the amino acid is an annotated functional sites

图11 Prophage-4末端酶大亚基系统发育树 采用最大似然法构建CR-HMKP K2-ST375 Prophage-4系统进化树，分支上的数字为自展值

Fig. 11 Phylogenetic tree of Prophage-4 based on the terminase large subunit The maximum likelihood method was used to construct the CR-HMKP K2-ST375 Prophage-4 phylogenetic tree, and the numbers on the branches were the bootstrap value

图12 Prophage-4 内溶素系统发育树 采用最大似然法构建CR-HMKP K2-ST375 Prophage-4编码的endolysin系统进化树，分支上的数字为自展值

Fig. 12 Phylogenetic tree of Prophage-4 based on the endolysin The maximum likelihood method was used to construct the endolysin phylogenetic tree encoded by CR-HMKP K2-ST375 Prophage-4, and the numbers on the branches were the bootstrap value