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We present evidence that methylammonium is transported into cowpea Rhizobium sp. strain 32H1 cells by a membrane carrier whose natural substrate is ammonium. After growth in low (0.2%) oxygen, which is necessary for nitrogen fixation by these cells, respiring rhizobial cells took up [14C]methylammonium to high intracellular levels. Cells grown in atmospheric (21%) oxygen did not take up methylammonium. Uptake (transport plus metabolism) was maximal in cells harvested in the early stationary phase of batch culture and had a distinct pH optimum of 6.5 to 7.0. Uptake was inhibited by metabolic poisons that dissipate the proton motive force or inhibit ATP synthesis. Inhibition of uptake by ammonium and the counterflow phenomenon indicated that ammonium and methylammonium share a transport carrier. Of the methylammonium taken up, about 15% was accumulated to intracellular levels 20 times higher than those in the medium; most of the methylammonium was metabolized to gamma-N-methylglutamine.

The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.

The bacterium Caulobacter crescentus undergoes an asymmetric cell division resulting in the formation of two different daughter cells, a motile swarmer cell and a nonmotile stalked cell. These two cell types differ in their program of gene expression, their ability to replicate DNA, and the physical properties of their nucleoids. We show here that two genes, gyrB (encoding the gyrase B subunit) and orf-1, are specifically transcribed from the chromosome in the portion of the predivisional cell destined for the progeny stalked cell. This is in contrast to a subset of flagellar genes which are transcribed from the chromosome in the incipient swarmer portion of the predivisional cell. gyrB and orf-1 are within a newly identified cluster of genes involved in DNA replication and recombination, including dnaN and recF. The transcription of gyrB and orf1 occurs from the replication-competent chromosome in stalked and predivisional cells and is silenced in swarmer cells. We hypothesize that selective silencing of groups of genes in the chromosomes at the swarmer and stalked poles of the predivisional cell results in the different developmental programs and the difference in replicative ability of the two progeny cells.

The obligate aerobe Cowpea Rhizobium sp. strain 32H1 in axenic culture is able to fix N2 when grown under 0.2% O2 but not when grown under 21% O2. It was, therefore, of interest to investigate ATP synthesis in these cells grown under the two conditions. When respiring in buffers having pHs ranging from 6 to 8.5, cells grown under either O2 tension maintained an intracellular pH more alkaline than the exterior. The transmembrane chemical gradient of H+ (delta pH) was essentially the same under both conditions of growth, decreasing from ca. 90 mV at medium pH 6 to ca. 30 mV at pH 8.5. However, the transmembrane electrical gradient (delta psi) was significantly higher in cells grown under 21% O2 (150 to 166 mV) than in cells grown under 0.2% O2, the latter being 16 mV at pH 6 and increasing to 88 mV at pH 8.5. Therefore, the proton motive force of 21% O2-grown cells ranged from 237 mV at external pH 6 to 185 mV at pH 8.5, compared with a proton motive force of 114 to 121 mV in the 0.2% O2-grown cells. The cells grown in 0.2% O2 had the same proton motive force whether tested at 21 or at 0.2% O2. The phosphorylation potential, calculated from the intracellular ATP, ADP, and Pi concentrations, was 424 mV in the 21% O2-grown cells and 436 mV in the 0.2% O2-grown cells. Thus, the 21% O2-grown cells translocated 1.8 to 2.3 H+/ATP synthesized by the H+-ATPase, whereas the H+/ATP ratio for 0.2% O2-grown cells was 3.7 to 3.8.

In Caulobacter crescentus, asymmetry is generated in the predivisional cell, resulting in the formation of two distinct cell types upon cell division: a motile swarmer cell and a sessile stalked cell. These progeny cell types differ in their relative programs of gene expression and DNA replication. In progeny swarmer cells, DNA replication is silenced for a defined period, but stalked cells reinitiate chromosomal DNA replication immediately following cell division. The establishment of these differential programs of DNA replication may be due to the polar localization of DNA replication proteins, differences in chromosome higher-order structure, or pole-specific transcription. The best-understood aspect of Caulobacter development is biogenesis of the polar flagellum. The genes encoding the flagellum are expressed under cell cycle control predominantly in the predivisional cell type. Transcription of flagellar genes is regulated by a trans-acting hierarchy that responds to both flagellar assembly and cell cycle cues. As the flagellar genes are expressed, their products are targeted to the swarmer pole of the predivisional cell, where assembly occurs. Specific protein targeting and compartmentalized transcription are two mechanisms that contribute to the positioning of flagellar gene products at the swarmer pole of the predivisional cell.

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.

An Escherichia coli lac operon constitutive for lacY was subcloned into the EcoRI site of a wide-host-range plasmid of the Q incompatibility group, and the resulting recombinant plasmid was introduced into Tn5-generated Lac- mutants of Rhizobium meliloti. The R. meliloti transconjugants accumulated lactose about 1,000-fold, equivalent to a proton motive force of -170 to -180 mV, not significantly different from the values calculated from the distributions of weak acids and lipophilic cations.