In this article we will discuss about the genetics of nitrogen fixing microorganisms.
1. Bacterial Nodulation Genes and Regulation of nod Gene Expression:
In Rhizobium species, nodulation genes together with other symbiotic genes are located on large-plasmids (Sym plasmids). Sym plasmids vary from 50 to over 600 kb in R. leguminosarum bv. trifoli to 1200 to 1500 kb in R. meliloti. Nodulation genes, nod and nol genes, are classified as regulatory, common and host specific. Regulation of nod genes is controlled by the nodD gene, of which all rhizobia tested so far contain one or more copies.
In conjunction with plant flavonoids or other phenolic compounds, nodD proteins act as transcriptional activators of inducible nod genes. Different nodD proteins respond to specific plant signal molecules and, therefore, contribute to host-specificity of nodulation. Most nodD genes are constitutively expressed, other are auto-regulated.
The common nodABC genes are structurally and functionally conserved among Rhizobium, Bradyrhizobuim and Azorhizobium strains. The inactivation of these genes completely abolishes root-hair infection and nodule formation. Putative function of nodulation gene is given in Table 14.4.
Table 14.4 : Possible functions of rhizobial Nod proteins.
The nif gene products of K. pneumoniae and their function are given in Table 14.5.
2. Nif Genes and their Regulation:
(i) K. pneumoniae:
The N2 fixation (nif) genes are organized into a regulon of 17 genes, consisting of seven or eight operons each of which is transcribed into a single, usually polycistronic mRNA. Although only five of the gene products have been purified and properly characterized, functions have been assigned to all of the genes except for nifX and nifY.
Regulation of nif gene expression has two elements; an external system designated ntr and an internal system mediated by nif A and nifL. The ntr system responds to conditions of nitrogen starvation by activating genes that enable the organism to utilize ‘unusual’ nitrogen sources such as arginine, proline, and histidine as well as N2 itself, in the last case by switching on the nif genes.
The inter-relationships between external and internal regulation of the nif genes in K. pneumoniae and the conditions under which nitrogenase synthesis occurs, are summarised in Fig. 14.4.
Fig. 14.4 : Regulation of nif gene expression in Kiebsiella pneumonlae.
Actually, the ntrA gene product (NtrA) is a-factor of RNA polymerase which recognizes the nif and, other ntr – regulated genes. These promoters have a structure different from that of typical bacterial promoters. NtrA allows RNA polymerase to bind at the nif promoters and to initiate transcription there.
The ntrB gene product (NtrB) is an enzyme that functions both as a protein kinase and as a phosphatase, the substrate of which is NtrC (the ntrC gene product).
Whether kinase or phosphatase activity predominates depends upon the nitrogen status of the bacterium, and the consequence of this is that, under condition of starvation, NtrC-P acts as an activator of, among other operons, nifL and nif A. The nif A product is an activator of transcription of other nif genes, whilst the nifL product, in the presence of either intermediate concentrations of fixed nitrogen or O2, inactivate the nif A product, thereby preventing transcription of other nif genes.
(ii) In Cyanobacteria:
In heterocystous cyanobacteria, the acquisition of nitrogenase activity in response to nitrogen deficiency is accompanied by the differentiation of vegetative cells into a specialised structure called ‘heterocysts’. This process has been studied by Haselkom (1986).
All non-heterocystous cyanobacteria possess the genes nifH, nifD, nifK as a cluster which is similar to K.pneumoniae. In the DNA of vegetative cells of heterocystous cyanobacteria the gene nifK is separated from the genes nifD and nifH as observed by Haselkorn (1986).
During the differentiation the intervening DNA of about 11000 base pairs (11 kb) is excised as a circle resulting in a clustered nif|HDK operon as studied in Anabaena PCC 7120. This excision is catalysed by the product of a gene, xisA, located within the excised 11 kb region as shown in Fig. 14.5.
Fig. 15.5 : Rearrangement of nif genes in Anabaena PCC7120 during heterocyst differentiation.
A second rearrangement has been observed during heterocyst differentiation by Heselkorn et al. (1986). This occurs, in the region of nif S, a gene involved in K. pneumoniae. In this arrangement, a segment of DNA of approximately 50 kb is excised, again as a circle, and after this rearrangement an operon with the structure nifB: ORF -1: nifS: ORF-2 is formed as shown in Fig. 14.5.
The excision occurs between ORF-1 and nifS, which is not catalysed by the xis A product. It is interesting to note that no expression of the nif H gene from Anabaena variables was found when it was inserted with its promoter into K. pneumoniae which gave the evidence that nif gene promoters in Anabaena PCC 7120 differ in structure from both E. coli promoters and the nif promoters of K. pneumoniae.