Influence of tyrosine-kinase Wzc activity on colanic acid production in Escherichia coli K12 cells

Bacterial tyrosine-kinases have been demonstrated to participate in the regulation of capsule polysaccharides (CPS) and exopolysaccharides (EPS) production and export. However, discrepant data have been reported on the molecular mechanism responsible for this regulation depending on the bacterial species analyzed. Special attention was previously paid to the tyrosine-kinase Wzc_ca of Escherichia coli K-12, which is involved in the production of the exopolysaccharide, colanic acid, and autophosphorylates by using a cooperative two-step process. In this work, we took advantage of these observations to investigate in further detail the effect of Wzc_ca phosphorylation on the colanic acid production. First, it is shown that expression of the phosphorylated form of Wzc prevents production of colanic acid whereas expression of the non-phosphorylated form allows biosynthesis of this exopolysaccharide. However, we provide evidence that, in the latter case, the size distribution of the colanic acid polymer is less scattered than in the case of the wild-type strain expressing both phosphorylated and non-phosphorylated forms of Wzc. It is then demonstrated that colanic acid production is not merely regulated by an on/off mechanism and that, instead, both phosphorylated and non-phosphorylated forms of Wzc are required to promote colanic acid synthesis. Moreover, a series of data suggests that besides the involvement of phosphorylated and non-phosphorylated forms of Wzc in the production of colanic acid, two particular regions of this kinase play as such an important role in the synthesis of this exopolysaccharide: a proline-rich domain located in the N-terminal part of Wzc_ca, and a tyrosine cluster present in the C-terminal portion of the enzyme. Furthermore, considering that polysaccharides are known to facilitate bacterial resistance to certain environmental stresses, it is shown that the resistance of E. coli to desiccation is directly connected with the phosphorylation state of Wzc_ca.     

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

Insertion mutagenesis of the metJBLF gene cluster of Escherichia coli K-12: evidence for an metBL operon.

The effects of Mu or transposon 5 insertions on the expression of genes of the metJBLF cluster show that metB and metL form an operon, transcribed from metB to metL, and that metF and metJ are independently transcribed.     

nfi, the gene for endonuclease V in Escherichia coli K-12.

Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B. Demple and S. Linn, J. Biol. Chem. 257:2848-2855, 1982). Thus, it may be a versatile DNA repair enzyme. The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified. nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12. This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization. Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.     

Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli.

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon+ cells, increased 38-fold in lon+ cells carrying a Tn10::delta kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::delta kan insertion. Sequencing of both sides of the Tn10::delta kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.     

Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12.

Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated. The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E. coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC. The mutant phenotype was recessive and physiologically undiscernible.