Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3,5-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ⌬MSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the ⌬MSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ⌬MSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.Cyclic AMP (cAMP) is an important intracellular second messenger that is involved in several signaling pathways in both bacteria and eukaryotes (2, 5). Studies have shown a role for cAMP not only in physiological processes such as catabolite repression and sporulation (16) but also in the regulation of several virulence pathways, e.g., in Pseudomonas, Vibrio, and Candida species (1,17,19,40,41). The synthesis of cAMP is dependent on the activity of adenylyl cyclases, which can be membrane-bound or soluble proteins (7). The largely differing amino acid sequences of nucleotide cyclases allow their classification into six classes, among which the class III nucleotide cyclases have the widest phyletic distribution (7, 37).Adenylyl and guanylyl cyclases of the class III family are proteins that form head-to-tail dimers and generate either two (homodimeric enzymes) or one (heterodimeric enzymes) active site at the dimer interface (20). Nucleotide cyclases catalyze the conversion of ATP or GTP to cAMP or GMP, respectively, in a metal-dependent manner, and biochemical and structural studies have identified specific sequence motifs that are involved in substrate (ATP or GTP) or metal (Mg 2ϩ ) binding (20). In contrast to the limited domain compositions of mammalian nucleotide cyclases, the bacterial counterparts are often fused to a variety of domains, highlighting their ability to be regulated allostericall...

No abstract

Biofilms are important biological structures formed on most submerged aquatic surfaces. They comprise a unique niche wherein a community of microorganisms co-exist. The study was undertaken to identify the bacterial flora associated with biofilms formed on the surface of larval rearing tanks in a prawn hatchery. Surface swabs of two randomly chosen larval rearing tanks in a hatchery were taken regularly throughout the hatchery cycle and the bacterial loads were estimated. The counts varied from 1.8x103 to 4.3x104 CFU.cm-2 in Tank I and 5.1x103 to 3.5x104 CFU.cm-2 in Tank II. No significant difference was observed between Tank I and Tank II in respect of biofilm bacterial count when enumerated (p<0.05). The common bacterial genera isolated in both tanks comprised gram-negative bacteria including Pseudomonas, Vibrio and Aeromonas. Bacillus and other non-spore formers were the predominant gram-positive bacteria isolated. Bacteria associated with biofilms are more resistant to antibiotics and water sanitizers generally used in tank water treatment. Biofilms can be a reservoir of pathogens and bacteria such as Pseudomonas, Aeromonas and Vibrio isolated from the biofilms in this study may be potential pathogens of prawn larvae in hatcheries.