Identification of a Dehydrogenase Required for Lactose Metabolism inCaulobacter crescentus

Abstract

Caulobacter crescentus, which thrives in freshwater environments with low nutrient levels, serves as a model system for studying bacterial cell cycle regulation and organelle development. We examined its ability to utilize lactose (i) to gain insight into the metabolic capacities of oligotrophic bacteria and (ii) to obtain an additional genetic tool for studying this model organism, aiming to eliminate the basal enzymatic activity that hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal). Using a previously isolated transposon mutant, we identified a gene, lacA, that is required for growth on lactose as the sole carbon source and for turning colonies blue in the presence of X-gal. LacA, which contains a glucose-methanol-choline (GMC) oxidoreductase domain, has homology to the flavin subunit of Pectobacterium cypripedii's gluconate dehydrogenase. Sequence comparisons indicated that two genes near lacA, lacB and lacC, encode the other subunits of the membrane-bound dehydrogenase. In addition to lactose, all three lac genes are involved in the catabolism of three other ␤-galactosides (lactulose, lactitol, and methyl-␤-D-galactoside) and two glucosides (salicin and trehalose). Dehydrogenase assays confirmed that the lac gene products oxidize lactose, salicin, and trehalose. This enzymatic activity is inducible, and increased lac expression in the presence of lactose and salicin likely contributes to the induction. Expression of lacA also depends on the presence of the lac genes, implying that the dehydrogenase participates in induction. The involvement of a dehydrogenase suggests that degradation of lactose and other sugars in C. crescentus may resemble a proposed pathway in Agrobacterium tumefaciens.Caulobacter species inhabit diverse aquatic and soil environments, their ubiquitous distribution reflecting an ability to prosper despite low nutrient levels (47, 48). Unraveling the physiological adaptations that enable these aerobic chemoheterotrophs to thrive under nutrient-poor conditions will facilitate interpretation of microbial activities in oligotrophic environments, such as water bodies (which make up the majority of the earth's surface). Recent studies of Caulobacter have led to novel findings regarding the uptake and catabolism of various carbon sources (6,14,38,44,52). In addition, one particular member of the group, Caulobacter crescentus, has emerged as a prominent model for the study of bacterial cell cycle progression, asymmetric cell division, and organelle development (7,8,34,49). Although factors that regulate the C. crescentus cell cycle appear to respond to changes in metabolic states (3,22,35), details of the connection remain nebulous. Investigation of C. crescentus's metabolic capabilities can improve understanding of bacterial cell cycle control as well as the biogeochemical contributions of oligotrophs.We initiated an examination of C. crescentus's ability to utilize lactose for two reasons. (i) C. crescentus cells predominantly dwell in freshwater pond...

The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives

“…Whether these two proteins act together or sequentially on the substrates to form the 3-ketodisaccharide is also unsolved. As Arellano et al (40) have suggested for C. crescentus, thuA and -B could code for two subunits of this enzyme. A limited and delayed growth was observed in leucrose-grown cultures of At7023 complemented with thuA of S. meliloti (Fig.…”

“…We have also been consistently unsuccessful in our attempts to detect other known intermediates of trehalose breakdown, such as glucose-6-phosphate, in reaction mixtures containing trehalose and induced S. meliloti cells or cell extracts, through thin-layer chromatography (TLC), HPLC, or HPLC-MS (results not presented). Previous studies with the related alphaproteobacte-rium A. tumefaciens suggested that disaccharide utilization pathways involving dehydrogenase activity may lead to the formation of 3-ketosugars (33,40,41). A. tumefaciens has also been shown to possess the unique ability not only to oxidize sucrose into its 3-keto derivative but also to secrete this compound into the culture medium (41).…”

“…In S. meliloti, on the other hand, thuAB disruption abolishes growth in trehalose-containing medium as well (20), indicating that trehalose assimilation in S. meliloti occurs only via 3-ketotrehalose formation. Arellano et al have reported a dehydrogenase-dependent assimilatory pathway for trehalose in Caulobacter crescentus (40).…”

Identification of a U/Zn/Cu responsive global regulatory two‐component system in Caulobacter crescentus

“…To accomplish this, a plasmid‐borne translational P urcA ‐ lacZ fusion (NJH123; Hillson et al ., ) was amplified with primers P urcA _UR_F and P urcA _UR_R (Table S12), and a fragment containing the 517‐bp region downstream of the urcA stop codon was amplified from the chromosome using P urcA _DR_F and P urcA _DR_R. Both fragments were cloned into the HindIII and BamHI‐digested pNPTS138 using In‐Fusion cloning and integrated into the chromosome of a lacA mutant (Arellano et al ., ) or NA1000 using the double‐crossover allele replacement as described above, yielding strains DMP89 and DMP90, respectively (Table S11). For the transposon screen, DMP89 was electroporated with VMCS2:: Tn5Pvan (Christen et al ., ) and plated onto PYE agar plates containing 40 μg ml −1 Xgal, 25 μg ml −1 kanamycin and 0 or 40 μM ZnSO 4 , yielding a total of ∼60,000 colonies.…”

Large-scale chemical-genetics of the human gut bacteriumBacteroides thetaiotaomicron

“…50 51 One of the most well studied constituents of the human microbiota is the Gram-negative gut 52 anaerobe Bacteroides thetaiotaomicron, which is prevalent in a substantial fraction of the 53 world's population 8 . Pioneering work on the human microbiota revealed the broad range of 54 complex polysaccharides and simple carbohydrates that B. thetaiotaomicron can degrade and 55 ferment in the gut [9][10][11] . Subsequently, analyses enabled by the full genome sequence of B.…”