## 1 Introduction
Alternatively, it is possible that upon production, curdlan helps to form a biofilm which engulfs the colony and forms a nebulous matrix of a biofilm without any discrete individual cell encapsulation. Only the former scenario would justify the formal designation of curdlan as a capsule according to the literature [26]. As one of the goals to this study, I wanted to investigate curdlan production by _agrobacterium_ species, in particular to see whether curdlan was also deposited as a discrete capsule on this bacterium. A single molecule of curdlan can be quite large, consisting of up to 12000 polymerized glucose residues [27]. Its size, as well as the unique, unbranched (1\(\rightarrow\)3)-\(\beta\)-D-glucan structure confers upon curdlan its unique gelling and high water-retention properties. There have been many reviews of the large body of literature covering the chemistry and physico-chemistry of curdlan, its pharmacological and immunomoddulation activities, and its commercial applications [21; 27; 28; 29; 30; 31; 32]. Curdlan is widely used, for example, as an additive to cement (super-workable concrete) for the construction industry, an additive to tablet formulations in the pharmaceutical industry, as well as an antiviral agent in immunotherapy and numerous applications in tissue engineering, drug delivery, and other biomedical applications. Moreover, curdlan is one of the most desirable edible polysaccharides because it lacks any unpleasant taste and because of its versatile applications as a thickener and healthy fiber in the food industry. Unfortunately, the industrial use of curdlan is limited by its high cost of production. One way to reduce the cost of production is to develop higher curdlan-yielding strains through genetic engineering. Genetic engineering of _agrobacterium_ strains to increase curdlan production was another goal of this study. The genetic basis of curdlan production has been investigated using _agrobacterium_ sp. strain ATCC31749. Earlier studies found that the essential genes include a biosynthetic gene cluster, _crdASC_, and _crdR_, which codes for a transcription regulator [33; 34]. CrdSshows some similarity to beta-glyoxyl transferases with repetitive action patterns [34]. The extremely hydrophobic CrdS protein is thought to form a multimeric (possibly an octamer) complex located in the inner membrane. This location is stabilized by seven transmembrane helices and a central large hydrophilic cytoplasmic region which contains the substrate-binding and catalytic motif. The _crdASC_ genes form a gene cluster (i.e., all transcribed in the same direction) within 5 kb of the linear chromosome of _agrobacteriumium_. The upstream of the first gene (_crdA_) in the _crd_ cluster is a strong promoter, referred to as _crdP_[35] or Prcd. It was not known whether Prcd is the only promoter for the _crdASC_ cluster. Transposon insertions into any of the genes _crdA_, _crdS_, or _crdC_ result in the inability to produce curdlan. Loss of curdlan production by the insertion disruption of _crdA_ is interesting because of the implication that either _crdA_ is essential for curdlan production and/or because of the polar effect of disruption of transcription of the downstream gene _crdS_. The functions of the protein products of the genes _crdA_ and _crdC_ are not known. The gene _pssAG_, coding for a phosphatidylinositolserine synthase, also affects curdlan yield [33]. It is likely that specific phospholipids are important for the activity of the synthase complex in the inner membrane of this gram-negative bacterium [36]. In addition, curdlan production appears to be strictly governed by complex gene expression regulation [27; 35; 37]. Upon encountering the environmental triggers for curdlan production (e.g., low nitrogen and abundant sugars) the transcription of the _crdASC_ genes increases by almost 100-fold, and this depended upon phosphate accumulation and the stringent response (ppGpp) [37].