Aromatic compounds are abundant in the biosphere as components of the complex polymer lignin and as environmental pollutants. The bacterial biodegradation of structurally simple, readily degradable aromatic compounds has been studied with the expectation that this will facilitate work on more recalcitrant members of the group. As a result, much information has been obtained about the enzymology and molecular regulation of aerobic pathways of aromatic compound degradation (6,16,35,53). An aspect of aromatic biology that has received almost no attention, however, is the question of how bacteria sense and respond to the presence of aromatic compounds in their environment. Chemotaxis and transport are two physiological functions that operate in this capacity, and a number of studies have shown that aromatic acids such as benzoate and 4-hydroxybenzoate are strong chemoattractants for Pseudomonas putida, as well as a number of other species of gram-negative bacteria including Agrobacterium spp. and rhizobia (19,20,31,39,40). However, very little is known about the characteristics of the presumed receptor proteins that are responsible for initial attractant recognition. Similarly, although a number of studies have inferred the existence of specific transport systems for aromatic acids and related compounds, only a few detailed studies of aromatic compound permeases have been reported (1, 2, 14, 30), and no molecular analyses of bacterial genes that encode such proteins have been presented.

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.Bacterial chemotaxis has been studied in detail for Escherichia coli and Salmonella enterica serovar Typhimurium (35). Simple sugars, amino acids, and organic acids are chemoattractants for these enteric bacteria. Aromatic acids such as benzoate, 4-hydroxybenzoate, and salicylate are attractants for Pseudomonas putida PRS2000 (15). Recently, the soil bacterium P. putida G7 was reported to be attracted to the pollutant naphthalene (12,24,31). This expanded the range of organic compounds that are known to serve as bacterial chemoattractants to include aromatic hydrocarbons. However, nothing is known about chemotaxis towards other common aromatic hydrocarbons such as toluene and benzene. Five distinct pathways have been described for the aerobic degradation of toluene. All pathways are initiated with the oxidation of toluene, but five different oxidation products are formed (Fig. 1). P. putida F1 contains toluene 2,3-dioxygenase, an enzyme that oxidizes the aromatic ring of toluene, incorporating both atoms of molecular oxygen. After a dehydrogenation step, 3-methylcatechol is formed. This compound is further degraded via meta ring fission (8, 10, 11). P. putida PaW15 (a leucine auxotroph of strain mt-2) initiates degradation at the methyl group of toluene, eventually forming benzoate. Benzoate is converted to catechol, which is also degraded by a meta cleavage route (41). Strains that monooxygenate the aromatic ring of toluene have also be...

Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.

The circadian clock of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 imposes a global rhythm of transcription on promoters throughout the genome. Inactivation of any of the four known group 2 sigma factor genes (rpoD2, rpoD3, rpoD4, and sigC), singly or pairwise, altered circadian expression from the psbAI promoter, changing amplitude, phase angle, waveform, or period. However, only the rpoD2 mutation and the rpoD3 rpoD4 and rpoD2 rpoD3 double mutations affected expression from the kaiB promoter. A striking differential effect was a 2-h lengthening of the circadian period of expression from the promoter of psbAI, but not of those of kaiB or purF, when sigC was inactivated. The data show that separate timing circuits with different periods can coexist in a cell. Overexpression of rpoD2, rpoD3, rpoD4, or sigC also changed the period or abolished the rhythmicity of PpsbAI expression, consistent with a model in which sigma factors work as a consortium to convey circadian information to downstream genes.

Cyanobacteria such as Synechococcus elongatus PCC 7942 exhibit 24-h rhythms of gene expression that are controlled by an endogenous circadian clock that is mechanistically distinct from those described for diverse eukaryotes. Genetic and biochemical experiments over the past decade have identified key components of the circadian oscillator, input pathways that synchronize the clock with the daily environment, and output pathways that relay temporal information to downstream genes. The mechanism of the cyanobacterial circadian clock that is emerging is based principally on the assembly and disassembly of a large complex at whose heart are the proteins KaiA, KaiB, and KaiC. Signal transduction pathways that feed into and out of the clock employ protein domains that are similar to those in two-component regulatory systems of bacteria.