Cloning and expression in Pseudomonas putida of two of the histidine utilization genes from Rhizobium fredii.

Two of the genes encoding histidine utilization (hut) in Rhizobium fredii strain HH303 have been cloned in Pseudomonas putida and partially characterized. Molecular cloning of the genes was achieved by mobilizing an R. fredii cosmid library into a mutant strain of P. putida containing a Tn5 element in its histidase (hutH) gene. A number of overlapping clones were identified, all of which contain a 7.1-kbp HindIII fragment. The origin of this 7.1-kbp fragment from the chromosome of R. fredii was confirmed by Southern blotting and hybridization studies. In addition, this fragment and the two adjacent HindIII fragments of 9 and 12 kbp respectively were subcloned into pBluescript KS+ and further characterized. Although the R. fredii clones expressed histidase in Pseudomonas, they did not in Escherichia coli, suggesting that E. coli is not a suitable cloning host for Rhizobia genes.

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.

RNA polymerase as a repressor of transcription in the hut(P) region of mutant Klebsiella aerogenes histidine utilization operons.

Mutants of Klebsiella aerogenes able to express the hutUH operon in the absence of positive effectors were isolated and characterized. These mutations improve the hutUH promoter (PUH) by changing the -10 region to match the consensus sequence more closely. These mutations also affect another, oppositely oriented promoter in this region, PC. Although the mutations lie far outside PC, they cause PC to be inactive, apparently because binding of RNA polymerase to the PUH promoter blocks the overlapping PC site. Thus, in the mutants, RNA polymerase bound at the strong (mutant) PUH site effectively repress the PC promoter.

Bidirectional promoter in the hut(P) region of the histidine utilization (hut) operons from Klebsiella aerogenes.

The hut(P) region (i.e., the region responsible for regulation of hutUH expression) of the Klebsiella aerogenes histidine utilization (hut) operons contains a bidirectional promoter. One transcript from this promoter encodes the hutUH operon; the role of the oppositely directed transcript is unknown, although it appears to be involved in regulating hutUH expression (A.J. Nieuwkoop, S.A. Boylan, and R.A. Bender, J. Bacteriol. 159:934-939, 1984). A 247-base-pair (bp) fragment containing hut(P) carries two RNA-polymerase-binding sites agree with the start sites of the two transcripts produced from hut(P) DNA in vitro and in vivo. The binding sites share a 4-bp region, suggesting that occupancy of the regulatory site precludes occupancy of the hutUH promoter, and vice versa. In the absence of positive effectors, the binding to the site responsible for hutUH transcription is weaker than the binding to the site responsible for regulation. The nucleotide sequence of the 250-bp fragment containing hut(P) contains two possible matches to the consensus sequence for Escherichia coli promoters, a better and worse match, corresponding in position to the stronger and weaker RNA-polymerase-binding sites, respectively. The sequence also contains a region similar to the consensus sequence for binding of the catabolite gene activator protein of E. coli. A sequence similar to the consensus for Ntr-dependent promoters was also found, overlapping both RNA-polymerase-binding sites, but it is not a functional promoter. Finally, an initiation codon preceded by a Shine-Dalgarno consensus sequence and followed by an open reading frame identifies a probable start of the hutU gene coding sequences.

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation.

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three non-auxotrophic Nod- mutants and one His-Nod- mutant of USDA110. Homogenotization of the cloned fragments into wild-type strain USDA110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.

Regulation of hutUH operon expression by the catabolite gene activator protein-cyclic AMP complex in Klebsiella aerogenes.

RNA polymerase transcribed the hutUH operon of Klebsiella aerogenes if the catabolite gene activator protein (CAP) and cyclic AMP (cAMP) were present or if the DNA template was derived from a promoter mutant in which hutUH expression was independent of the need for positive effectors. In the absence of CAP or cAMP, not only was hutUH transcription absent, but transcription in the opposite direction (toward hutC) was initiated at a site (pC) ca. 70 base pairs from the site (pUH) of hutUH mRNA initiation. When the pC promoter was cloned in front of a promoterless galK gene, active expression of galK was observed. Thus, the pC promoter is active in vivo as well as in vitro. Transcription from pUH and pC may be mutually exclusive, with the major effect of CAP and cAMP being to prevent transcription from pC, thus relieving the antagonistic effect on transcription from pUH. This "double-negative" control by CAP-cAMP is supported by two observations: (i) CAP-cAMP was unable to activate transcription from pUH if RNA polymerase had been previously bound to pC and (ii) a mutation that allowed transcription from pUH in the absence of positive effectors simultaneously eliminated the activity of pC. An alternative model, in which CAP-cAMP is required for pUH expression and RNA polymerase binding at pC serves to modulate this control in some unknown way, is also considered. The physiological role of the transcript from pC other than regulation of pUH is unknown.