No abstract

The fixation of atmospheric N 2 by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena , this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N 2 fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N 2 fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF + motif, a palindrome at or close to position −35 that seems essential for heterocyst-specific expression of certain genes.

The efficiency of conjugal transfer of plasmids from Escherichia coli to the cyanobacterium Anabaena sp. strain PCC 7120 was quantitated as a function of the number of restriction sites for the restriction enzymes carried by the recipient. In addition to the previously recognized isoschizomers of AvaI and AvaII, PCC 7120 was found to possess an isoschizomer of AvaIII. Plasmids modified in E. coli with methylases that protect in vitro against restriction by the three enzymes were transferred with high efficiency, nearly independent of the number of restriction sites on the plasmid. Plasmids left unprotected against one of the three restriction enzymes were transferred with lower efficiencies. For low numbers of sites, the efficiency of conjugal transfer decreased as an exponential function of the number of unprotected sites. The methods presented may be used to increase the efficiency of conjugal transfer into restriction-competent bacteria.Since first recognized, restriction in vivo has been most closely identified with the reduction of plating efficiency of bacteriophage (7, 10). Although conjugal transfer of unmodified DNA also was shown to be sensitive to restriction in Escherichia coli (10), the inability of certain restriction enzymes to act on single-stranded DNA (49, 56) has led to the view that conjugally transferred single-stranded DNA (37) should be immune from restriction (5, 69). Accordingly, there are many examples in which conjugal transfer is unaffected by restriction barriers that prevent the infection of unmodified phage or the transformation of unmodified DNA (5,20,36,41,55,66). However, there are also reports of restriction in nonenteric bacteria indeed limiting conjugal efficiency (18,44,64,79).Regardless of the biological importance of restriction, the resulting inefficiency of DNA transfer is a frequently encountered barrier against introducing DNA into bacteria of experimental interest. While restriction may reduce the frequency of DNA transfer below levels of detectability (40,42,79), it is also possible that some introduced DNA with unmodified restriction sites may escape destruction. With this in mind, it is often of practical importance to know quantitatively the degree to which each restriction site impairs DNA transfer into restriction-competent recipients.The effect of restriction sites on the efficiency of DNA transfer has been quantitated in several studies using plasmids or phage with a known number of sites. Such studies have measured the effect of restriction on phage infection (47, 48, 54), phage transfection (35), transformation (17,51,71), and electroporation (8,34,43). Reduction of conjugal transfer efficiency by restriction has not been systematically studied, although there have been some quantitative reports (30,70 (14,18,42). We report here the construction of plasmids carrying methylases that protect against one or more restriction activities of Anabaena sp. strain PCC 7120, including a third restriction activity reported here for the first time. Using these plasmids, ...