摘要
进入21世纪以来,能源危机、环境污染的问题越来越突出,影响到了人类的健康生存和可持续发展。发展可持续的、绿色能源来代替石油、煤炭等化石资源已经成为人们迫切的需求。以木质纤维素为主要成分的植物生物质是地球上最丰富的可再生资源,对木质纤维素的开发利用越来越受到人们的重视。由于纤维素自身结构致密,并存在结晶区,不容易被酶解,使得现有的纤维素生物转化效率低,成本高。因此揭示自然界中高效的纤维素降解机制,提高结晶纤维索的降解效率是木质纤维素利用的关键。
Cytophaga hutchinsonii是一种自然界中广泛分布的纤维素降解细菌,具有极强的结晶纤维素降解能力。C. hutchinsonii不分泌游离的纤维素酶也没有纤维小体结构,表明其具有不同于已知的好氧真菌或厌氧细菌的独特的结晶纤维素降解机制。此外,C. hutchinonii的不依赖于鞭毛或纤毛的滑动机制也是未解之谜。C. hutchinsonii降解纤维素时,菌体有序地排列在纤维素纤维上,研究者猜测Chutchinsonii的滑动与纤维素降解有关。对C. hutchinsonii新型结晶纤维素降解机制的研究能够丰富人们对微生物纤维素降解策略的认识,并有助于对现有纤维素降解酶系的改进和提高,促进纤维素资源的开发利用。之前由于缺少成熟的遗传操作体系,对C. hutchinsonii纤维素降解与滑动的研究进展十分缓慢。近几年我们实验室初步建立C. hutchinsonii的部分遗传操作技术,为纤维素降解及滑动机制的研究深入到分子水平奠定了基础。本文首次建立了Himai转座子的插入突变和基因回补技术,为基因功能的研究创造了条件。利用这些技术首次筛选到多个参与C. hutchinsonii纤维素降解与滑动的关键基因,并分别对其基因功能进行了深入研究。在此基础上发现了C. hutchinsonii存在细胞表面的纤维素利用途径,并对菌体运动与纤维素降解之间的关系进行了探讨。这些研究工作对揭示Chutchinsonii独特的纤维素降解和滑动机制有着重要意义。具体内容和结果如下：
1. Himar转座子插入突变和基因回补技术的建立
转座子插入突变是研究基因功能的有效手段。我们尝试了两种转座子Tn4351和HimarEm3对C. hutchinsonii (?)勺转座插入突变,转座子HimarEm3改造自HimarEM1。发现在接合平板中加入20μg/ml的Km可以将接合转化效率提高一倍,通过优化接合转化条件,两种转座子的转化率分别达到3.0×10和1.2×10-6。对Tn4351在F. Johnsonicae(?)中的转座插入研究发现其插入形式比较复杂多样。我们通过Southern blot分析了HimarEm3在C. hutchinsonii中的插入情况,发现其大多情况下是单插入形式,这表明HimarEm3是很好的插入失活研究基因功能的工具。
红霉素是已知的在C. hutchinsanii中唯一可用的抗生素筛选标记,筛选标记的缺少限制了C. hutchinsonii中的遗传操作。通过实验发现头孢西丁抗性基因(cfxA)和四环素抗性基因(tetQ)能够在C. hutchinsonii中表达。以此为筛选标记构建了可用于回补转座子Tn4351和HimarEm3的插入突变的质粒载体,为转座插入研究基因功能创造了条件。
2. CHU_0134基因功能研究
菌体对纤维素的吸附是其酶解的第一步,序列分析发现C. hutchinsonii(?)编码的纤维素酶大都不含CBM,菌体与纤维素直接接触是纤维素降解所必需的。我们通过转座子HimarEm3的插入突变筛选到一株菌体对纤维素的吸附率只有野生菌株一半的突变株A-4。表型测定发现A-4不能降解纤维素并表现出琼脂表面菌落扩散缺陷。纤维素酶活测定发现突变株细胞表面及胞外的内切纤维素酶酶活下降40%。纤维素吸附外膜蛋白的SDS-PAGE电泳发现,突变株中大部分的纤维素吸附外膜蛋白含量降低或消失。质谱鉴定差异蛋白条带为一些Gld蛋白(CHU0171、0173、0174、3494)和一些未知功能蛋白(CHU_3654、1277、3655)。这些蛋白可能在C. hutchinsonii的纤维素吸附、降解中起重要作用。
Southern blot与反向PCR确定A-4中HimarEm3的插入基因为CHU_0134。回补基因CHU0134(?)使突变株的纤维素吸附、降解及琼脂表面的菌落扩散表型恢复。研究结果表明CHU_0134能够影响C. hutchinsonii(?)菌体的纤维素吸附、降解以及在琼脂表面的菌落扩散。序列分析预测CHU0134编码一个硫醇-二硫化物异构酶,在C-端有包含C-G-H-G的类TlpA结构。硫醇-二硫化物异构酶可以催化蛋白质中二硫键的异构作用,对于很多细胞质以外的蛋白质的结构以及稳定性是非常重要的。序列比对发现拟杆菌中有很多蛋白与CHU0134有较高的序列相似性,而且大都含有保守的C-G-H-C序列,这类蛋白的功能值得研究。
3.CHU1277基因功能研究
CHU1277编码一个纤维素吸附外膜蛋白,我们通过基因的定点插入失活构建了CHU_1277失活突变株(△1277),纤维索吸附外膜蛋白中CHU_1277蛋白条带缺失证明CHU_1277被失活。RT-PCR显示△1277中CHU_1277的临近基因的转录没有受到影响。△1277表现出纤维索利用缺陷以及纤维寡糖利用能力的降低,说明CHU_1277是影响C. hutchinsonii (?)纤维素类底物利用的关键基因。
△1277的内切纤维素酶酶活与野生菌株相比没有明显变化,菌体表面β-葡萄糖苷酶酶活下降30%。HPLC分析菌体对纤维二糖的水解发现A1277菌体的纤维二糖水解能力明显低于野生菌株,与△1277利用纤维二糖的生长很弱相吻合。然而提取的△1277的外膜蛋白却表现出与野生菌株相似的β-葡萄糖苷酶酶活和纤维二糖水解能力。这表明突变株细胞表面的β-葡萄糖苷酶并没有减少,只是活力没有充分展现出来。细胞表面蛋白CHU_1277对位于细胞表面的β-葡萄糖苷酶的酶活充分发挥可能是必需的。
提取的突变株的外膜蛋白有着与野生菌株基本相同的纤维素酶酶活以及纤维素水解能力,表明突变株外膜蛋白中的纤维素酶的量与野生菌株基本相同,纤维素酶并不是导致突变株纤维素降解缺陷的主要原因。这暗示着只有纤维素酶对C. hutchinsonii的纤维素降解还是不够的,某些非酶蛋白的参与是纤维素降解所必需的。CHU_1277是发现的第一个影响C. hutchinsonii纤维素降解的表面蛋白。深入探索CHU_1277如何影响C. hutchinsonii的纤维素降解将对阐明其独特的纤维素降解机制有着重要意义。
4. C. hutchinsonii对纤维二糖的利用
纤维二糖是已知的C. hutchinsonii可以利用的少数几种寡糖之一,又是纤维素降解中的重要中间产物。我们首次研究了C. hutchinsonii(?)寸纤维二糖的利用方式。虽然基因组序列分析预测可能的β-葡萄糖苷酶都位于细胞周质,但是多次酶活实验表明大部分的β-葡萄糖苷酶活力位于细胞表面。菌体对纤维二糖的水解实验也表明C. hutchinsonii的细胞表面有很强的纤维二糖水解能力,目前正在对细胞表面的β-葡萄糖苷酶进行研究。
HPLC分析C. hutchinsonii在纤维二糖培养基中生长时培养基中的糖分变化发现,接种之后,培养基中的纤维二糖就被逐渐水解,葡萄糖逐渐积累,在Chulchinsonii达到对数生长中期之前,培养基中的纤维二糖已经基本被完全水解,积累了很高浓度的葡萄糖,葡萄糖成为支持细胞对数生长中期之后继续生长的唯一碳源。这表明通过细胞表面的β-葡萄糖苷酶将纤维二糖水解成葡萄糖再吸收利用是C. hutchinsonii利用纤维二糖的主要途径。
5. C. hutchinsonii细胞表面的纤维素降解
HPLC分析菌体对纤维索的水解产物发现一部分的纤维素可以在Chutchinsonii的细胞表面被降解。通过加入葡萄糖酸内酯抑制β-葡萄糖苷酶酶活以及加入NaN3(?)抑制细胞的呼吸产能,发现细胞表面的纤维素降解是先由内切纤维素酶将纤维素水解为纤维寡糖(纤维二糖、三糖、四糖),然后再由β-葡萄糖苷酶将纤维寡糖水解为葡萄糖。提取的C. hutchinsonii的外膜蛋白中有内切纤维素酶酶活和β-葡萄糖苷酶酶活,能够在体外水解纤维素,葡萄糖是唯一产物,说明纤维素在外膜蛋白中的内切纤维素酶和β-葡萄糖苷酶的共同作用下能够被水解为葡萄糖,这也支持纤维素细胞表面降解。
纤维素细胞表面降解不符合Wilson提出C. hutchinsonii的纤维素降解模型,模型中预测纤维素链从纤维素上被剥离下来,然后运到细胞周质中进一步被内切纤维素酶降解。在我们的实验中,在胞外只检测到很少量的纤维寡糖产物,因此尚不能确定纤维素的细胞表面降解是C. hutchinsonii利用纤维素的主要方式。但是在C. hutchinsonii的细胞表面的确存在纤维素降解过程,并且降解产物可以被菌体吸收。而C. hutchinsonii是以何种形式吸收纤维索水解产物的还有待进一步的研究。
6. CHU_1797基因功能研究
我们通过转座子HimarEm3的插入突变以及基因回补确证了一个新的影响C. hutchinsonii在琼脂表面菌落扩散的基因,(CHU_1797。序列分析预测CHU_1797编码一个包含未知功能结构域DUF3308的外膜蛋白。比对发现与CHU_1797有序列相似性的蛋白广泛分布于拟杆菌中,大都含有未知功能结构域DUF3308,预测大都定位在膜上。这类膜蛋白可能与菌体的运动有关,有待进一步研究。CHU1797失活突变株在琼脂表面的菌落扩散出现缺陷,进一步影响了菌体在琼脂农面趋向葡萄糖的运动以及与埋在琼脂中的纤维素底物的接触。但是CHU1797的失活并不影响单个菌体在玻璃表面上的滑动,突变株对纤维索的降解能力也没有明显变化。扫描电镜观察发现,突变株菌体在纤维素纤维上排列有序,与野生菌株的现象相同。这说明C. hutchinsonii在琼脂表面的菌落扩散能力不是菌体在玻璃表面的个体运动以及纤维索降解所必需的。C. hutchinsonii(?)琼脂表面的菌落扩散与在玻璃表面的个体运动是两种不同的运动形式,存在着不同的机制,而单个菌体在玻璃表面的运动可能与菌体在纤维素纤维上的有序排列相关。
Since the beginning of twenty-first century, the problems of energy crisis and environmental pollution are increasingly serious and seriously threaten human's health and sustainable development. Exploration sustainable green energy to replace oil, coal and other fossil resources has become the urgent need. Lignocellulose, the main component of plant biomass, is the most abundant renewable resource on the earth. More and more attention was paid to the utilization of lignocellulose. However, cellulose is difficult for enzymatic degradation due to its compact structure and the presence of crystalline region. At present, low efficiency and high cost are general problem in cellulose bioconversion. Exploitation the efficient cellulose degradation mechanisms in the nature to resolve the efficient degradation of crystalline cellulose is the key of the lignocellulose utilization.
Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that can rapidly digest crystalline cellulose using a novel strategy different from most aerobic fungi and anaerobic bacteria. It does not secrete soluble extracellular cellulolytic enzymes and has no cellulosome-like structure. Besides, the mechanism of its cell motility over surfaces without flagella and type IV pili is not known. C. hutchinsonii cells align themselves along cellulose fiber regularly when they digest them suggesting cell motility might facilitate cellulose digestion. The study of the novel mechanism of crystalline cellulose degradation by C. hutchinsonii would improve the understanding of the microbial cellulose utilization strategies, and also conducive to take advantage of cellulosic biomass. However, due to the lack of genetic manipulation tools, few study focused on the cellulose degradation and gliding mechanisms of C. hutchinsonii. In this study, gene functional study in C. hutchinsonii through transposon mutagenesis and complementation was first developed, and these genetic techniques provided an opportunity to understand the details of the novel cellulose degradation and gliding mechanisms of C. hutchinsonii. Several functional genes involved in cellulose degradation and gliding were further studied, and the utilization of cellulosic substrates of C. hutchinsonii was also discussed.
1. Development of transposon mutagenesis and complementation
Transposon mutagenesis is effective to study gene function. In this study, two kinds of transposons, Tn4351and HimarEm3, were translocated into C. hutchinsonii through conjugation. HimarEm3was derived from HimarEm1. The transformation frequency would be increased with addition of20μg/ml kanamycin in conjugation medium, and transformation frequency of3.0×10-7or1.2×10-6was finally achieved for Tn4351or HimarEm3, respectively. Complicated insertion of Tn4351in F. johnsoniae was reported. The insertion of HimarEm3in C. hutchinsonii was studied by southern blot, and the results showed that most transformants had a single insertion in the genome. This demonstrated that transposon HimarEm3was an beneficial and convenient tool to study gene function in C. hutchinsonii.
The erythromycin resistance gene was the only reported selective marker gene used in C. hutchinsonii, which was an limitation for further genetic manipulation in C. hutchinsonii. In order to find more selective genes functional in C. hutchinsonii, several antibiotic resistance genes were tried to express in C. hutchinsonii. The results showed that cfxA and tetO were functional in C. hutchinsonii. The complemented plasmids carrying cfxA or tetQ for erythromycin insertion were constructed, and gene functional study through transposon mutagenesis and complementation was available.
2. Functional study of CHU_0134
Direct contact with the cellulose was necessary for effective cellulose degradation by C. hutchinsonii. However, genomic analysis showed that C. hutchinsonii does not possess cellulosome, and most of the encoded cellulase has no carbohydrate-binding module (CBM). The mutant A-4with significantly reduced cellulose binding ability was constructed through transposon mutagenesis The relative adhesion rate of the mutant was only a half of that of the wild-type strain. The mutant A-4was deficient in cellulose degradation and colony spreading on agar. Cellulase assay showed that the endocellulase activity of the cell-free supernatants and on the intact cell surface of A-4decreased by40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4including some Gld proteins (CHU_0171,0173,0174) and hypothetical proteins (CHU_3654,1277,3655). It was speculated that these proteins might play an important role in cellulose binding and degradation by C. hutchinsonii.
The site of HimarEm3insertion of A-4was analyzed by Southern blot and inverse PCR, and the results showed that the transposon was inserted in gene CHU_0134. The CHU_0134complement strain could restore cell motility, cellulose adhesion and degradation to nearly the level of the wild-type strain indicating that these phenotypic defects of the A-4was caused by mutation in CHU_0134. CHU_0134is annotated to encode a thiol-disulfide isomerase since there was a putative TlpA-like family/thioredoxin (TRX)-like super-family domain containing a Cys-X-X-Cys motif (C369-G-H-C372) in its C-terminal. Thiol-disulfide isomerase can catalyze the isomerization of disulfide bonds in protein which are important for the structure and stability of many extra-cytoplasmic proteins. Sequence analysis of proteins with a sequence similar to CHU0134showed that the most of these proteins belong to bacteria in the phylum Bacteroidetes with nearly the same conserved motif C-G-H-C, and the function of these proteins needs further study.
3. Functional study of CHU_1277
CHU_1277encoded a cellulose binding outer membrane protein, and inactived by insertional mutagenesis. The SDS-PAGE of cellulose binding outer membrane protein revealed that CHU_1277was absent in the CHU_1277disrupted mutant, indicating that CHU_1277was inactived. The result of RT-PCR showed that gene transcription of the adjacent genes of CHU_1277was not affected. The disruption of CHU_1277caused drastic effect on the growth parameters on cellulosic substrate. The mutant failed to digest cellulose and its oligosaccharides utilization ability was significantly reduced, indicating that CHU_1277was essential for cellulose degradation and played an important role in cellooligosaccharide utilization by C. hutchinsonii.
Cellulase assay showed that endocellulase activity of the mutant was almost the same as that of the wild-type strain, while β-glucosidase activity on the cell surface was decreased by30%. The cellobiose hydrolytic activity of the mutant cells was significant reduced according to the analysis of the cellobiose hydrolysis of the intact cells, which was consistent with the poor growth of the mutant in the cellobiose medium. However, the outer membrane proteins extracted from the mutant cells exhibited similar β-glucosidase activity and cellobiose hydrolytic activity to that of the wild-type strain in vitro. These results implied that the quantity and activity of β-glucosidase on the mutant cell surface was actually unreduced, and low activity was exhibited on the cell surface may be caused by the disruption of CHU1277. We speculated that the direct or indirect interaction with CHU_1277might be necessary for the cell surface β-glucosidases to achieve sufficient activity.
The study of cellulase activity and cellulose hydrolytic activity of the outer membrane proteins suggested that the cellulolytic enzymes of the mutant were almost integral. This implied that cellulolytic enzymes were not sufficient for cellulose utilization by C. hutchinsonii. Some other non-enzymatic proteins were necessary for effective cellulose degradation. CHU_1277was the first cell-surface protein proved to be essential for cellulose degradation by C. hutchinsonii, and further study of the function of CHU_1277in cellulose degradation would help to clarify the novel mechanism of cellulose degradation by C. hutchinsonii.
4. Cellobiose utilization of C. hutchinsonii
Cellobiose is one of the few substrates that can be used by C. hutchinsonii as the sole carbon and energy source, which is also the main intermediate product of cellulose degradation. The cellobiose utilization of C. hutchinsonii was first studied. Most of the β-glucosidase activity was located on the cell surface of C. hutchinsonii from the enzyme assay, although the genomic analysis showed that all the candidate β-glucosidases were predicted to be located in the periplasmic space. Native PAGE proved there was an active β-glucosidase band in the sample of outer membrane proteins of C. hutchinsonii, and significant cellobiohydrolase activity was detected on the cell surface and in the outer membrane proteins of C. hutchinsonii.
After inoculation of C. hutchinsonii in cellobiose medium, cellobiose was gradually hydrolyzed and glucose was accumulated. Cellobiose in the culture was almost completely hydrolyzed before the mid-exponential phase, and glucose was accumulated to a high concentration. Then, glucose was the sole carbon source to provide further growth of the bacterium. This indicated that cellobiose was hydrolyzed into glucose by β-glucosidases on the cell surface was the main way of cellobiose utilization by C. hutchinsonii.
5. Cellulose degradation on the cell surface of C. hutchinsonii
We first detected that cellulose could be hydrolyzed to glucose on the cell surface of C. hutchinsonii through the analysis of cellulose hydrolyzate by intact cells. With addition of glucono-δ-lactone or NaN3to repress β-glucosidase activity or inhibit energy production through the respiratory chain reaction, we found the process of cellulose degradation on the cell surface of C. hutchinsonii. Cellulose was first hydrolyzed to cellooligosaccharides by cell-surface endocellulases, and cellooligosaccharides could be further hydrolyzed to glucose by cell-surface glucosidases. The extracted outer membrane proteins containing free cellulases were also proved to have ability to hydrolyze cellulose in vitro with glucose as the only product, indicating that cellulose would be hydrolyzed to glucose with the role of endocellulases and β-glucosidases in the outer membrane.
Cellulose degradation on the cell surface was different from the possible model proposed by wilson that individual cellulose molecules were removed from cellulose fibers by an outer membrane protein complex and transported into the periplasmic space, then degraded by endoglucanases there. Transient accumulation of cellooligosaccharides was detected in cellulose degradation by intact cells leading to the doubt whether it is the main way of cellulose utilization of C. hutchinsonii. Nevertheless, cellulose could be hydrolyzed on the cell surface and cellulosic hydrolyzate also could be assimilated by the cells of C. hutchinsonii. Assimilation and translocation of cellulose hydrolyzate of the C. hutchinsonii cells need to be further studied.
6. Functional study of CHU_1797
Transposon mutagenesis and complementation were used to identify a new locus, CHU_1797, essential for colony spreading on agar surfaces. CHU_1797encodes a putative outer-membrane protein of348amino acids with unknown function. Proteins which have high sequence similarity to CHU_1797were widespread in the members of the phylum Bacteroidetes, almost had a DUF3308domain and located on the membrane from the protein subcellular localization prediction. These proteins might be related to gliding motility, and this speculation needs more experimental evidence.
The disruption of CHU_1797suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. The gliding of the mutant cells on the glass surface was not affected, and the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild-type strain from the SEM observation. These results indicated that the colony spreading ability on agar surfaces was not required for gliding of the C. hutchinsonii cells on the glass surface and cellulose degradation. This implied that the mechanism of spreading and orderly arrangement of cells on the surface of cellulose fiber was different from the mechanism underlying colony spreading over agar surfaces, but may be related to the motility of individual cells over wet glass surfaces.
引文
Agarwal S, Hunnicutt DW, McBride MJ (1997) Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc Natl Acad Sci U S A 94 (22):12139-12144
Alvarez B, Secades P, McBride MJ, Guijarro JA (2004) Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol 70 (1):581-587
Alvarez B, Secades P, Prieto M, McBride MJ, Guijarro JA (2006) A mutation in Flavobacterium psychrophilum tlpB inhibits gliding motility and induces biofilm formation. Appl Environ Microbiol 72 (6):4044-4053
Aubert JP, Beguin P, Millet J, Federation of European Microbiological Societies.. Societe francaise de microbiologie. (1988) Biochemistry and genetics of cellulose degradation. FEMS symposium, vol no 43. Academic Press, London; San Diego
Baker MD, Wolanin PM, Stock JB (2006) Signal transduction in bacterial chemotaxis. Bioessays 28 (1):9-22
Bayer EA, Coutinho PM, Henrissat B (1999) Cellulosome-like sequences in Archaeoglobus fulgidus. an enigmatic vestige of cohesin and dockerin domains. FEBS Lett 463 (3):277-280
Bayer EA, Morag E, Lamed R (1994) The cellulosome--a treasure-trove for biotechnology. Trends in biotechnology 12 (9):379-386
Bayer EA, Shimon LJ, Shoham Y, Lamed R (1998) Cellulosomes-structure and ultrastructure. J Struct Biol 124 (2-3):221-234
Beguin P, Lemaire M (1996) The cellulosome:an exocellular, multiprotein complex specialized in cellulose degradation. Crit Rev Biochem Mol Biol 31 (3):201-236
Bhaya D, Bianco NR, Bryant D, Grossman A (2000) Type Ⅳ pilus biogenesis and motility in the cyanobacterium Synechocystis sp. PCC6803. Mol Microbiol 37 (4):941-951
Braun TF, Khubbar MK, Saffarini DA, McBride MJ (2005) Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis. J Bacteriol 187(20):6943-6952
Braun TF, McBride MJ (2005) Flavobacterium johnsoniae GldJ is a lipoprotein that is required for gliding motility. J Bacteriol 187 (8):2628-2637
Carvalho AP, Fernandes PA, Ramos MJ (2006) Similarities and differences in the thioredoxin superfamily. Prog Biophys Mol Biol 91 (3):229-248
Chang LE, Pate JL, Betzig RJ (1984) Isolation and characterization of nonspreading mutants of the gliding bacterium Cytophaga johnsonae. J Bacteriol 159 (1):26-35
Chang WT, Thayer DW (1977) The cellulase system of a Cytophaga species. Can J Microbiol 23 (9):1285-1292
Chen H, Huang N, Sun Z (2006) SubLoc:a server/client suite for protein subcellular location based on SOAP. Bioinformatics 22 (3):376-377
Chen N, Xu Y, Wang H, Lu X (2012) Factors affecting adhesion of Cytophaga hutchinsonii to cellulose. Wei Sheng Wu Xue Bao 52 (8):1027-1032
Cheng PT, Pritzker KP, Kandel RA, Reid A (1983) Analytical scanning and transmission electron microscopy and x-ray microdiffractometry of calcium pyrophosphate dihydrate crystal deposits in tissues. Scan Electron Microsc (Pt 1):369-377
Cherry JR, Fidantsef AL (2003) Directed evolution of industrial enzymes:an update. Current Opinion in Biotechnology 14 (4):438-443
Costerton JW, Irvin RT, Cheng KJ (1981) The bacterial glycocalyx in nature and disease. Annu Rev Microbiol 35:299-324
Ding SY, Bayer EA, Steiner D, Shoham Y, Lamed R (2000) A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type 11 cohesins. J Bacteriol 182 (17):4915-4925
Ding SY, Xu Q, Crowley M, Zeng Y, Nimlos M, Lamed R, Bayer EA, Himmel ME (2008) A biophysical perspective on the cellulosome:new opportunities for biomass conversion. Curr Opin Biotechnol 19 (3):218-227
Doyle RJ, Sonnenfeld EM (1989) Properties of the cell surfaces of pathogenic bacteria. Int Rev Cytol 118:33-92
Dutton RJ, Boyd D, Berkmen M, Beckwith J (2008) Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation. Proc Natl Acad Sci U S A 105 (33):11933-11938
Gardy JL, Spencer C, Wang K, Ester M, Tusnady GE, Simon I, Hua S, deFays K, Lambert C, Nakai K, Brinkman FS (2003) PSORT-B:Improving protein subcellular localization prediction for Gram-negative bacteria. Nucleic Acids Res 31 (13):3613-3617
Glavina Del Rio T, Abt B, Spring S, Lapidus A, Nolan M, Tice H, Copeland A, Cheng JF, Chen F, Bruce D, Goodwin L, Pitluck S, Ivanova N, Mavromatis K, Mikhailova N, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Chang YJ, Jeffries CD, Chain P, Saunders E, Detter JC, Brettin T, Rohde M, Goker M, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Kyrpides NC, Klenk HP, Lucas S (2010) Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand Genomic Sci 2 (1):87-95
Gleiter S, Bardwell JC (2008) Disulfide bond isomerization in prokaryotes. Biochim Biophys Acta 1783 (4):530-534
Gillian W, Reese E T. Evidence for multiple components in microbial cellulase. Can J Microbial,1984,17(1):90-107
Gong J, Forsberg CW (1989) Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose. Appl Environ Microbiol 55 (12):3039-3044
Gong J, Forsberg CW (1993) Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase. J Bacteriol 175 (21):6810-6821
Han C, Spring S, Lapidus A, Del Rio TG, Tice H, Copeland A, Cheng JF, Lucas S, Chen F, Nolan M, Bruce D, Goodwin L, Pitluck S, Ivanova N, Mavromatis K, Mikhailova N, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Chang YJ, Jeffries CC, Saunders E, Chertkov 0, Brettin T, Goker M, Rohde M, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Kyrpides NC, Klenk HP, Detter JC (2009a) Complete genome sequence of Pedobacter heparinus type strain (HIM 762-3). Stand Genomic Sci 1 (1):54-62
Han W, Xu Z, Su J, Lu X, Gao P (2009b) Morphologic changes in the life cycle of Cytophaga hutchinsonii. Wei Sheng Wu Xue Bao 49 (1):18-22
Harshey RM (1994) Bees aren't the only ones:swarming in gram-negative bacteria. Mol Microbiol 13 (3):389-394
Hartzell PL, Youderian P (1995) Genetics of gliding motility and development in Myxococcus xanthus. Arch Microbiol 164 (5):309-323
Henrichsen J (1983) Twitching motility. Annu Rev Microbiol 37:81-93
Henrichsen J, Blom J (1975) Examination of fimbriation of some gram-negative rods with and without twitching and gliding motility. Acta Pathol Microbiol Scand B 83(3):161-170
Henrissat B, Driguez H, Viet C, Schulein M (1985) Synergism of Cellulases from Trichoderma reesei in the Degradation of Cellulose. Nat Biotech 3 (8):722-726
Hodgkin J, and Kaiser D (1979a). Genetics of gliding motility in Myxococcus xanthus (Myxobacterales):genes controlling movement of single cells. Mol Gen Genet 171,167-176
Hodgkin J, and Kaiser D (1979b). Genetics of Gliding Motility in Myxococcus xanthus(Myxobacterales):Two gene systems control movement. Mol Gen Genet 171,177-191
Hoiczyk E (2000) Gliding motility in cyanobacterial:observations and possible explanations. Arch Microbiol 174 (1-2):11-17
Hunnicutt DW, Kempf MJ, McBride MJ (2002) Mutations in Flavobacterium johnsoniae gldF and gldG disrupt gliding motility and interfere with membrane localization of GldA. J Bacteriol 184 (9):2370-2378
Hunnicutt DW, McBride MJ (2000) Cloning and characterization of the Flavobacterium johnsoniae gliding-motility genes gldB and gldC. J Bacteriol 182 (4):911-918
Hunnicutt DW, McBride MJ (2001) Cloning and characterization of the Flavobacterium johnsoniae gliding motility genes gldD and gldF. J Bacteriol 183 (14):4167-4175
James GT (1978) Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem 86 (2):574-579
Jarvis M (2003) Chemistry:cellulose stacks up. Nature 426 (6967):611-612.
Ji X, Xu Y, Zhang C, Chen N, Lu X (2012) A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii. Appl Microbiol Biotechnol 96(1):161-170
Jun HS, Qi M, Gong J, Egbosimba EE, Forsberg CW (2007) Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion. J Bacteriol 189 (19):6806-6815
Jung H, Wilson DB, Walker LP (2003) Binding and reversibility of Thermobifida fusca Cel5A, Cel6B, and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose. Biotechnol Bioeng 84 (2):151-159
Jung HQ United States. Agricultural Research Service., U.S. Dairy Forage Research Center. (1993) Forage cell wall structure and digestibility. American Society of Agronomy Crop Science Society of America Soil Science Society of America, Inc., Madison, Wis., USA
Kaiser D (1979) Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc Natl Acad Sci U S A 76 (11):5952-5956
Kaiser, D, and Crosby, C (1983). Cell movements and its coordination in swarms of Myxococcus xanthus. Cell Motil 3,275-284.
Kempf MJ, McBride MJ (2000) Transposon insertions in the Flavobacterium johnsoniae ftsX gene disrupt gliding motility and cell division. J Bacteriol 182 (6):1671-1679
Klein GL, Snodgrass WR.1993. Cellulose. In:Macrae R, Robinson RK, Saddler MJ, editors. Encyclopedia of food science, food technology and nutrition. London: Academic Press, p 758-767
Lamed R, Naimark J, Morgenstern E, Bayer EA (1987) Specialized cell surface structures in cellulolytic bacteria. J Bacteriol 169 (8):3792-3800
Larkin, J.M.1989. Nonphotosynthetic, nonfruiting gliding bacteria, p.2010-2138. In J. T. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol.3. Williams and Wilkins, Baltimore, MD.
Lehtio J, Sugiyama J, Gustavsson M, Fransson L, Linder M, Teeri TT (2003) The binding specificity and affinity determinants of family I and family 3 cellulose binding modules. Proceedings of the National Academy of Sciences of the United States of America 100 (2):484-489
Li Y, Sun H, Ma X, Lu A, Lux R, Zusman D, Shi W (2003) Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus. Proc Natl Acad Sci U S A 100 (9):5443-5448
Lindberg F, Lund B, Johansson L, Normark S (1987) Localization of the receptor-binding protein adhesin at the tip of the bacterial pilus. Nature 328 (6125):84-87
Linder M, Teeri TT (1996) The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose. Proc Natl Acad Sci U S A 93 (22):12251-12255
Louime C, Abazinge M, Johnson E (2006) Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cylophaga huichinsonii. International Journal of Molecular Sciences 7 (1):1-11
Louime C, Abazinge M, Johnson E, Latinwo L, Ikediobi C, Clark AM (2007) Molecular cloning and biochemical characterization of a family-9 endoglucanase with an unusual structure from the gliding bacteria Cytophaga hutchinsonii. Appl Biochem Biotechnol 141 (1):127-138
Luciano J, Agrebi R, Le Gall AV, Wartel M, Fiegna F, Ducret A, Brochier-Armanet C, Mignot T (2011) Emergence and modular evolution of a novel motility machinery in bacteria. PLoS Genet 7 (9):e1002268
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66 (3):506-577, table of contents
Lynd LR, Wyman CE, Gerngross TU (1999) Biocommodity Engineering. Biotechnol Prog 15(5):777-793
Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EG, Grigoriev IV, Harris P, Jackson M, Kubicek CP, Han CS, Ho I, Larrondo LF, de Leon AL, Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B, Salamov AA, Schmoll M, Terry A, Thayer N, Westerholm-Parvinen A, Schoch CL, Yao J, Barabote R, Nelson MA, Detter C, Bruce D, Kuske CR, Xie G, Richardson P, Rokhsar DS, Lucas SM, Rubin EM, Dunn-Coleman N, Ward M, Brettin TS (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 26 (5):553-560
Mattick JS (2002) Type Ⅳ pili and twitching motility. Annu Rev Microbiol 56:289-314
McBride MJ (2001) Bacterial gliding motility:multiple mechanisms for cell movement over surfaces. Annu Rev Microbiol 55:49-75
McBride MJ, Baker SA (1996) Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga. Appl Environ Microbiol 62 (8):3017-3022
McBride MJ, Braun TF (2004) Gldl is a lipoprotein that is required for Flavobacterium johnsoniae gliding motility and chitin utilization. J Bacteriol 186 (8):2295-2302
McBride MJ, Braun TF, Brust JL (2003) Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization. J Bacteriol 185(22):6648-6657
McBride MJ, Kempf MJ (1996) Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. J Bacteriol 178 (3):583-590
McBride MJ, Rhodes RG, Pochiraju S, Bollampalli S (2009) Nevel motility and protein secretion machinery of Flavobacterium johnsoniae. Available online.
McBride MJ, Zhu Y (2013) Gliding motility and Por secretion system genes are widespread among members of the phylum bacteroidetes. J Bacteriol 195 (2):270-278
Mignot T (2007) The elusive engine in Myxococcus xanthus gliding motility. Cell Mol Life Sci 64(21):2733-2745
Mignot T, Shaevitz JW, Hartzell PL, Zusman DR (2007) Evidence that focal adhesion complexes power bacterial gliding motility. Science 315 (5813):853-856
Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry 31 (3):426-428
Miron J, Ben-Ghedalia D, Morrison M (2001) Invited review:adhesion mechanisms of rumen cellulolytic bacteria. J Dairy Sci 84 (6):1294-1309
Morrison M, Pope PB, Denman SE, McSweeney CS (2009) Plant biomass degradation by gut microbiomes:more of the same or something new? Curr Opin Biotechnol20(3):358-363
Nelson SS, Bollampalli S, McBride MJ (2008) SprB is a cell surface component of the Flavobacterium johnsoniae gliding motility machinery. J Bacteriol 190 (8):2851-2857
Nelson SS, McBride MJ (2006) Mutations in Flavobacterium johnsoniae secDF result in defects in gliding motility and chitin utilization. J Bacteriol 188 (1):348-351
Nupur, Sharma S, Kumar Singh P, Suresh K, Anil Kumar P (2012) Fulvivirga imtechensis sp. nov., a member of the phylum Bacteroidetes. Int J Syst Evol Microbiol 62 (Pt 9):2213-2217
Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120 (3):621-623
Pagani I, Chertkov O, Lapidus A, Lucas S, Del Rio TG, Tice H, Copeland A, Cheng JF, Nolan M, Saunders E, Pitluck S, Held B, Goodwin L, Liolios K, Ovchinikova G, Ivanova N, Mavromatis K, Pati A, Chen A, Palaniappan K, Land M, Hauser L Jeffries CD, Detter JC, Han C, Tapia R, Ngatchou-Djao OD, Rohde M, Goker M, Spring S, Sikorski J, Woyke T, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Klenk HP, Kyrpides NC (2011) Complete genome sequence of Marivirga tractuosa type strain (H-43). Stand Genomic Sci 4 (2):154-162
Peter Z (2001) Conformation and packing of various crystalline cellulose fibers. Progress in Polymer Science 26 (9):1341-1417
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0:discriminating signal peptides from transmembrane regions. Nat Methods 8 (10):785-786
Rhodes RG, Samarasam MN, Shrivastava A, van Baaren JM, Pochiraju S, Bollampalli S, McBride MJ (2010) Flavobacterium johnsoniae gldN and gldO are partially redundant genes required for gliding motility and surface localization of SprB. J Bacteriol 192 (5):1201-1211
Rhodes RG, Samarasam MN, Van Groll EJ, McBride MJ (2011) Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion. J Bacteriol 193 (19):5322-5327
Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, Mekalanos JJ (1999) In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc Natl Acad Sci U S A96 (4):1645-1650
Sato K, Naito M, Yukitake H, Hirakawa H, Shoji M, McBride MJ, Rhodes RG, Nakayama K (2010) A protein secretion system linked to bacteroidete gliding motility and pathogenesis. Proc Natl Acad Sci U S A 107 (1):276-281
Shi W, Zusman DR (1993) The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces. Proc Natl Acad Sci U S A 90 (8):3378-3382
Shouldice SR, Heras B, Walden PM, Totsika M, Schembri MA, Martin JL (2011) Structure and function of DsbA, a key bacterial oxidative folding catalyst. Antioxid Redox Signal 14 (9):1729-1760
Shrivastava A, Rhodes RQ Pochiraju S, Nakane D, McBride MJ (2012) Flavobacterium johnsoniae RemA is a mobile cell surface lectin involved in gliding. J Bacteriol 194 (14):3678-3688
Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175-182
Spormann AM (1999) Gliding motility in bacteria:insights from studies of Myxococcus xanihus. Microbiol Mol Biol Rev 63 (3):621-641
Stanier RY (1942) The Cytophaga Group:A Contribution to the Biology of Myxobacteria. Bacteriol Rev 6 (3):143-196
Staroscik AM, Hunnicutt DW, Archibald KE, Nelson DR (2008) Development of methods for the genetic manipulation of Flavobacterium columnare. BMC Microbiol 8:115
Su H, Shao Z, Tkalec L, Blain F, Zimmermann J (2001) Development of a genetic system for the transfer of DNA into Flavobacterium hepavinum. Microbiology 147 (Pt 3):581-589
Sun M, Wartel M, Cascales E, Shaevitz JW, Mignot T (2011) Motor-driven intracellular transport powers bacterial gliding motility. Proc Natl Acad Sci U S A 108 (18):7559-7564
Thomas S, Russell JB (2004) The effect of cellobiose, glucose, and cellulose on the survival of Fibrobacter succinogenes A3C cultures grown under ammonia limitation. Curr Microbiol 48 (3)219-223
Tomme P, Warren RA, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1-81
Uenoyama A, Kusumoto A, Miyata M (2004) Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass binding during gliding of Mycoplasma mobile. J Bacteriol 186 (5):1537-1545
Urban A, Leipelt M, Eggert T, Jaeger KE (2001) DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa. J Bacteriol 183 (2):587-596
Walker E, Warren FL (1938) Decomposition of cellulose by Cytophaga. Ⅰ. Biochem J 32 (1):31-43
Wall D, Kaiser D (1999) Type Ⅳ pili and cell motility. Mol Microbiol 32 (1):1-10
Wang L, Zhang Y, Gao P, Shi D, Liu H, Gao H (2006) Changes in the structural properties and rate of hydrolysis of cotton fibers during extended enzymatic hydrolysis. Biotechnol Bioeng 93 (3):443-456
Ward MJ, Zusman DR (1997) Regulation of directed motility in Myxococcus xanthus. Mol Microbiol 24 (5):885-893
Weon HY, Kim BY, Yoo SH, Lee SY, Kwon SW, Go SJ, Stackebrandt E (2006) Niastella koreensis gen. nov., sp. nov. and Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. Int J Syst Evol Microbiol 56 (Pt 8):1777-1782
Wilson DB (2008) Three microbial strategies for plant cell wall degradation. Ann N Y Acad Sci 1125:289-297
Wilson DB (2009) Evidence for a novel mechanism of microbial cellulose degradation. Cellulose 16 (4):723-727
Wolgemuth CW, Mogilner A, Oster G (2004) The hydration dynamics of polyelectrolyte gels with applications to cell motility and drug delivery. Eur Biophys J 33 (2):146-158
Wolgemuth CW, Oster G (2004) The junctional pore complex and the propulsion of bacterial cells. J Mol Microbiol Biotechnol 7 (1-2):72-77
Wu SS, Kaiser D (1995) Genetic and functional evidence that Type Ⅳ pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol 18 (3):547-558
Xia ZJ, Wang J, Hu W, Liu H, Gao XZ, Wu ZH, Zhang PY, Li YZ (2008) Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics. J Ind Microbiol Biotechnol 35 (10):1157-1163
Xie Q Bruce DC, Challacombe JF, Chertkov O, Detter JC, Gilna P, Han CS, Lucas S, Misra M, Myers GL, Richardson P, Tapia R, Thayer N, Thompson LS, Brettin TS, Henrissat B, Wilson DB, McBride MJ (2007) Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii. Appl Environ Microbiol 73 (11):3536-3546
Xu Y, Ji X, Chen N, Li P, Liu W, Lu X (2011) Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii. Appl Microbiol Biotechnol
Yomo T, Yamano T, Yamamoto K, Urabe I (1997) General Equation of Steady-State Enzyme Kinetics Using Net Rate Constants and Its Applicaiton to the Kinetic Analysis of Catalase Reaction. Journal of Theoretical Biology 188 (3):301-312
Youderian P (1998) Bacterial motility:secretory secrets of gliding bacteria. Curr Biol 8(12):R408-411
Yu NY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ, Brinkman FS (2010) PSORTb 3.0:improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes.
Zhang Y, Ducret A, Shaevitz J, Mignot T (2012) From individual cell motility to collective behaviors:insights from a prokaryote, Myxococcus xanthus. FEMS Microbiol Rev 36 (1):149-164
Zhang YH, Cui J, Lynd LR, Kuang LR (2006) A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7 (2):644-648
Zhang YH, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose:noncomplexed cellulase systems. Biotechnol Bioeng 88 (7):797-824
Zhang YH, Hong J, Ye X (2010) Cellulase Assays Biofuels. In:Mielenz JR (ed), vol 581. Methods in Molecular Biology. Humana Press, pp 213-231
Zhu Y, Li H, Zhou H, Chen G, Liu W (2010) Cellulose and cellodextrin utilization by the cellulolytic bacterium Cytophaga hutchisonii. Bioresour Technol 101 (16):6432-6437
Zhu Y, Zhou H, Bi Y, Zhang W, Chen G, Liu W (2012) Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchimonii. Appl Microbiol Biotechnol
Zusman DR, McBride MJ (1991) Sensory transduction in the gliding bacterium Myxococcus xanthus. Mol Microbiol 5 (10):2323-2329