BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole

Biofilms are microbial communities characterized by three-dimensional growth resulting from the ability of individual cells to adhere to each other as well as to produce an extracellular matrix that ensures biofilm physical cohesion. Numerous bacteria produce cellulose as a biofilm matrix polymer, a property relying on the expression of bacterial cellulose synthesis (Bcs) proteins and their post-translational activation upon binding of cyclic di-guanosine mono-phosphate second messenger (c-di-GMP) produced by diguanylate cyclases. In Escherichia coli and other Enterobacteriaceae, two genes of unknown function, yhjR and yhjQ , are located upstream of the bcs genes. Here, we show that yhjQ , but not yhjR , is essential for cellulose biosynthesis; it has therefore been renamed bcsQ. Using a green fluorescent protein (GFP) fusion approach, we demonstrate that BcsQ, a MinD homologue, displays a polar localization and that cell-to-cell adhesion is initiated through production of cellulose at the BcsQ-labelled pole. Although we did not detect a similar localization for other Bcs proteins, immunogold labelling of cellulose itself at the pole of individual bacteria indicates the localized activity of the cellulose biosynthesis apparatus. These results therefore suggest that BcsQ could participate in spatial restriction of cellulose biosynthesis activity in Enterobacteriaceae.     

A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon.

Recently, it was shown that a cellulose-negative mutant (Cel1) of Acetobacter xylinum ATCC 23769 carried an insertion of an indigenous transposable element (IS1031A) about 500 bp upstream of the bcs operon, required for cellulose synthesis. Here we show that Cel1 can be complemented by wild-type DNA covering the insertion point. Nucleotide sequencing of this region revealed the presence of two open reading frames, ORF1 and ORF2. ORF2, which is disrupted by the IS1031A insertion in Cel1, potentially encodes the complementing function. ORF1 encodes a protein (CMCax) with significant homology to previously described endoglucanases. A cloned DNA fragment containing ORF1 expressed a carboxymethyl cellulose-hydrolyzing activity in Escherichia coli. In A. xylinum, CMCax is secreted into the culture growth medium. The CMCax mature protein consists of 322 amino acids and has a molecular mass of 35.6 kDa.     

A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis.

We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level.     

Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon.

Two enzymes, S-adenosylmethionine decarboxylase and spermidine synthase, are essential for the biosynthesis of spermidine in Escherichia coli. We have previously shown that the genes encoding these enzymes (speD and speE) form an operon and that the area immediately upstream from the speE gene is necessary for the expression of both the speE and speD genes. We have now studied the upstream promoter and the downstream terminator regions of this operon more completely. We have shown that the major mRNA initiation site (Ia) of the operon is located 475 base pairs (bp) upstream from the speE gene and that there is an open reading frame that encodes for a polypeptide of 115 amino acids between the Ia site and the ATG start codon for the speE gene. Downstream from the stop codon for the speD gene is a potential hairpin structure immediately followed by an mRNA termination site, t. An additional mRNA termination site, t', is present about 110 bp downstream from t and is stronger than t. By comparing our DNA fragments with those prepared from this region of the E. coli chromosome by Kohara et al., we have located the speED operon on the physical map of the E. coli chromosome. We have shown that the orientation of the speED operon is counterclockwise and that the operon is located 137.5 to 140 kbp (2.9 minutes) clockwise from the zero position of the E. coli chromosomal map.     

Differential mRNA stability controls relative gene expression within a polycistronic operon

In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon. The polycistronic malEFG operon of E. coli contains two REP sequences (highly conserved inverted repeats) within the malE-malF intercistronic region. Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein. A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA. The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common.     

Resistin and RELM-alpha gene expression in white adipose tissue of lactating mice

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.     

The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein.

We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpl3 gene. This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E. coli. We have shown that the rpl3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon. However, no typical procaryotic promoter could be found for this gene. We have detected, using anti E. coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E. coli cells transformed with the cyanelle rpl3 gene.     

Occurrence of mazEF-like antitoxin/toxin systems in bacteria

The mazEF locus of Escherichia coil located in an operon together with the upstream relA gene (encoding ATP:GTP 3'-pyrophosphotransferase; (p)ppGpp synthetase), encodes an antitoxin/toxin system which might play a role in programmed cell death under stress and starvation conditions at high cell densities. By homology searches, chromosomally encoded orthologous systems were identified in a variety of bacteria, sometimes without the MazE-like antitoxin, and several bacterial species possess multiple MazEF-like systems (paralogs). In many gram positive bacteria, the mazEF-locus is located directly upstream of the sigB (stress sigma factor sigmaB) operon in a putative operon together with the upstream dal (aIr) gene (encoding D-alanine racemase). The acidic antitoxins are less conserved than the basic toxins. The differences in genomic organization of the mazEFloci in E. coli versus those in gram positive bacteria might indicate their association with different stress response regulons in these organisms. A study on the sigmaB operon of Staphylococcus aureus showed that the mazF gene of this organism is cotranscribed with the sigmaB operon in response to heat shock, providing the first example that the expression of the mazEFlocus might be indeed associated with stress responses.     

Structural characterization of the Salmonella typhimurium LT2 umu operon.

The umuDC operon of Escherichia coli encodes functions required for mutagenesis induced by radiation and a wide variety of chemicals. The closely related organism Salmonella typhimurium is markedly less mutable than E. coli, but a umu homolog has recently been identified and cloned from the LT2 subline. In this study the nucleotide sequence and structure of the S. typhimurium LT2 umu operon have been determined and its gene products have been identified so that the molecular basis of umu activity might be understood more fully. S. typhimurium LT2 umu consists of a smaller 417-base-pair (bp) umuD gene ending 2 bp upstream of a larger 1,266-bp umuC gene. The only apparent structural difference between the two operons is the lack of gene overlap. An SOS box identical to that found in E. coli is present in the promoter region upstream of umuD. The calculated molecular masses of the umuD and umuC gene products were 15.3 and 47.8 kilodaltons, respectively, which agree with figures determined by transpositional disruption and maxicell analysis. The S. typhimurium and E. coli umuD sequences were 68% homologous and encoded products with 71% amino acid identity; the umuC sequences were 71% homologous and encoded products with 83% amino acid identity. Furthermore, the potential UmuD cleavage site and associated catalytic sites could be identified. Thus the very different mutagenic responses of S. typhimurium LT2 and E. coli cannot be accounted for by gross differences in operon structure or gene products. Rather, the ability of the cloned S. typhimurium umuD gene to give stronger complementation of E. coli umuD77 mutants in the absence of a functional umuC gene suggests that Salmonella UmuC protein normally constrains UmuD protein activity.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.