Sixteen nif and 'nif-associated genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp. strain RF-1 have been cloned and sequenced. All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons. The nifH operon (nifH-nifD-nifK) has been reported previously. nifB, fdXN, nifS8 nifU and nifP were found to be located upstream of the nifH operon. nifB-WxN-nifS-nifU were expressed as an operon. A nifPlike gene was found to be located just upstream of nifB. nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and 'fdx' were found to be located downstream from nifK. The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB-' fdx' and span approximately 7 kb. The function of the ORF situated between nifX and niNV is not known. However, it was identified as a counterpart of ORF-2 in Anabaena sp. strain PCC 7120 based on the deduced amino acid sequence. Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifx-orf, niW-hesA-hesB and fdx'-containing operons, respectively. According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h IighVl2 h dark regimen. The rhythm persisted after the culture was transferred to continuous illumination.

The typical citrus canker lesions produced by Xanthomonas axonopodis pv. citri are erumpent, callus-like, with water-soaked margins. Three novel atypical symptom-producing variants of X. axonopodis pv. citri were described recently in Taiwan. Only the variant designated as A(f) type produces typical erumpent canker lesions on Mexican lime (Citrus aurantifolia) but induces flat necrotic with water-soaked margin lesions on grapefruit leaves (C. paradisi). Two homologous pthA were cloned and characterized from strains XW19 (a typical canker lesion producing strain) and XW47 (a strain of A(f) type). The pthA homolog from XW19 was transformed into XW47. The transformant of XW47 induced typical erumpent canker lesions on grapefruit leaves. Sequence analyses of transformants XW19 and XW47 revealed over 99% homology in nucleotide and deduced amino acid sequences compared with pthA homologs deposited in GenBank. The amino acid residues located at positions 49, 286, 742 and 767 of PthA were different between XW47 and XW19. The PthA mutants with a single amino acid substitution at each of these four positions were constructed by site-directed mutagenesis. Modified PthA (S286P) from XW47 in transformant 47SP induced erumpent canker lesions on grapefruit leaves, whereas another modified PthA (P286S) from XW19 in transformant 47PS only induced flat necrotic lesions. These results suggested that a single amino acid substitution from either serine to proline or proline to serine at position 286 of PthA can alter canker formation by X. axonopodis pv. citri on grapefruit leaves

Erwinia chrysanthemi (Ech) RA3B produces a large amount of blue indigoidine. Using Tn5-induced mutagenesis, three indigoidine-deficient mutants were generated. Followed by library screening, a 5.8kb fragment complemented mutants for indigoidine synthesis was cloned. This fragment contains four complete open-reading frames (ORFs), pecS, pecM, idgA, and idgB, and two partial ORFs, argG, and idgC. These genes are nearly identical to those in strain Ech3937. Primer extension assays demonstrated a clear transcriptional start site prior to idgA, while no promoter preceding idgB and idgC was detected, suggesting that idgA, idgB, and idgC are organized as one transcription unit. In contrast, indAB is separated from indC in Ech3937. Interestingly, an ERIC sequence was present between idgB and idgC in place of the promoter region of the homolog indC, which may contribute to the loss of promoter activity in RA3B. Futhermore, idgB mutant displayed much lighter blue color, while indB mutant appeared white on media. Overexpression of pecS in RA3B resulted in significantly reduced indigoidine production and idgC transcript. Moreover, gel shift and luxAB reporter assays revealed that PecS specifically binds to the sequence preceding idgA and inhibits gene expression, which is consistent with the results observed in Ech3937.