Weaver J, Whitehead T R, Cotta M A, Valentine P C, Salyers A A
Department of Microbiology, University of Illinois, Urbana 61801.
Appl Environ Microbiol. 1992 Sep;58(9):2764-70. doi: 10.1128/aem.58.9.2764-2770.1992.
Bacteroides ovatus, a gram-negative obligate anaerobe found in the human colon, can utilize xylan as a sole source of carbohydrate. Previously, a 3.8-kbp segment of B. ovatus chromosomal DNA, which contained genes encoding a xylanase (xylI) and a bifunctional xylosidase-arabinosidase (xsa), was cloned, and expression of the two genes was studied in Escherichia coli (T. Whitehead and R. Hespell, J. Bacteriol. 172:2408-2412, 1990). In the present study, we have used segments of the cloned region to construct insertional disruptions in the B. ovatus chromosomal locus containing these two genes. Analysis of these insertional mutants demonstrated that (i) xylI and xsa are probably part of the same operon, with xylI upstream of xsa, (ii) the true B. ovatus promoter was not cloned on the 3.5-kbp DNA fragment which expressed xylanase and xylosidase in E. coli, (iii) there is at least one gene upstream of xylI which could encode an arabinosidase, and (iv) xylosidase rather than xylanase may be a rate-limiting step in xylan utilization. Insertional mutations in the xylI-xsa locus reduced the rate of growth on xylan, but the concentration of residual sugars at the end of growth was the same as that with the wild type. Thus, a slower rate of growth on xylan was not accompanied by less extensive digestion of xylan. Mutants in which xylI had been disrupted still expressed some xylanase activity. This second activity was associated with membranes and produced xylose from xylan, whereas the xylI gene product partitioned primarily with the soluble fraction and produced xylobiose from xylan.
卵形拟杆菌是一种在人类结肠中发现的革兰氏阴性专性厌氧菌，它能够利用木聚糖作为唯一的碳水化合物来源。此前，已克隆出卵形拟杆菌染色体DNA的一个3.8 kbp片段，该片段包含编码木聚糖酶（xylI）和双功能木糖苷酶-阿拉伯糖苷酶（xsa）的基因，并在大肠杆菌中研究了这两个基因的表达（T.怀特黑德和R.赫斯佩尔，《细菌学杂志》172:2408 - 2412，1990）。在本研究中，我们利用克隆区域的片段在卵形拟杆菌染色体位点构建了包含这两个基因的插入性破坏。对这些插入性突变体的分析表明：（i）xylI和xsa可能是同一个操纵子的一部分，xylI在xsa的上游；（ii）在大肠杆菌中表达木聚糖酶和木糖苷酶的3.5 kbp DNA片段上没有克隆到卵形拟杆菌真正的启动子；（iii）xylI上游至少有一个基因可能编码阿拉伯糖苷酶；（iv）木糖苷酶而非木聚糖酶可能是木聚糖利用中的限速步骤。xylI - xsa位点的插入性突变降低了在木聚糖上的生长速率，但生长结束时残留糖的浓度与野生型相同。因此，在木聚糖上生长较慢并不伴随着木聚糖消化程度的降低。xylI被破坏的突变体仍表达一些木聚糖酶活性。这种第二种活性与细胞膜相关，能从木聚糖产生木糖，而xylI基因产物主要分布在可溶性部分，能从木聚糖产生木二糖。
